In the present study, we have demonstrated functional interaction between Ste20-related proline-alanine-rich kinase (SPAK), WNK4 [with no lysine (K)], and the widely expressed Na+-K+-2Cl- cotransporter type 1 (NKCC1). NKCC1 function, which we measured in Xenopus laevis oocytes under both isosmotic (basal) and hyperosmotic (stimulated) conditions, was unaffected when SPAK and WNK4 were expressed alone. In contrast, expression of both kinases with NKCC1 resulted in a significant increase in cotransporter activity and an insensitivity to external osmolarity or cell volume. NKCC1 activation is dependent on the catalytic activity of SPAK and likely also of WNK4, because mutations in their catalytic domains result in an absence of cotransporter stimulation. The results of our yeast two-hybrid experiments suggest that WNK4 does not interact directly with NKCC1 but does interact with SPAK. Functional experiments demonstrated that the binding of SPAK to WNK4 was also required because a SPAK-interaction-deficient WNK4 mutant (Phe997Ala) did not increase NKCC1 activity. We also have shown that the transport function of K+-Cl- cotransporter type 2 (KCC2), a neuron-specific KCl cotransporter, was diminished by the expression of both kinases under both isosmotic and hyposmotic conditions. Our data are consistent with WNK4 interacting with SPAK, which in turn phosphorylates and activates NKCC1 and phosphorylates and deactivates KCC2.

To better understand why sensory neurons express voltage-gated Na+ channel isoforms that are different from those expressed in other types of excitable cells, we compared the properties of the hNE sodium channel [a human homolog of PN1, which is selectively expressed in dorsal root ganglion (DRG) neurons] with that of the skeletal muscle Na+ channel (hSkM1) [both expressed in human embryonic kidney (HEK293) cells]. Although the voltage dependence of activation was similar, the inactivation properties were different. The V1/2 for steady-state inactivation was slightly more negative, and the rate of open-state inactivation was approximately 50% slower for hNE. However, the greatest difference was that closed-state inactivation and recovery from inactivation were up to fivefold slower for hNE than for hSkM1 channels. TTX-sensitive (TTX-S) currents in small DRG neurons also have slow closed-state inactivation, suggesting that hNE/PN1 contributes to this TTX-S current. Slow ramp depolarizations (0.25 mV/msec) elicited TTX-S persistent currents in cells expressing hNE channels, and in DRG neurons, but not in cells expressing hSkM1 channels. We propose that slow closed-state inactivation underlies these ramp currents. This conclusion is supported by data showing that divalent cations such as Cd2+ and Zn2+ (50-200 microM) slowed closed-state inactivation and also dramatically increased the ramp currents for DRG TTX-S currents and hNE channels but not for hSkM1 channels. The hNE and DRG TTX-S ramp currents activated near -65 mV and therefore could play an important role in boosting stimulus depolarizations in sensory neurons. These results suggest that differences in the kinetics of closed-state inactivation may confer distinct integrative properties on different Na+ channel isoforms.

High blood lactate concentration (hyperlactacidaemia) in trauma or sepsis is thought to indicate tissue hypoxia and anaerobic glycolysis even when blood pressure, cardiac output, and urine output are within clinically acceptable ranges. However, mechanisms of lactate generation by well-oxygenated tissues have received little attention. Within cells, oxidative and glycolytic energy production can proceed in separate, independent compartments. In skeletal muscle and other tissues, aerobic glycolysis is linked to ATP provision for the Na+-K+ pump, the activity of which is stimulated by epinephrine. In injured patients, hypokalaemia may reflect increased Na+,K+-ATPase activity. We propose that increased blood lactate often reflects increased aerobic glycolysis in skeletal muscle secondary to epinephrine-stimulated Na+,K+-ATPase activity and not anaerobic glycolysis due to hypoperfusion. The hypothesis explains why hyperlactacidaemia often neither correlates with traditional indicators of perfusion nor diminishes with increased oxygen delivery. When other variables have returned to normal, continued attempts at resuscitation based on elevated blood lactate may lead to unnecessary use of blood transfusion and inotropic agents in an effort to increase oxygen delivery and lactate clearance.

Local, rhythmic, subsarcolemmal Ca2+ releases (LCRs) from the sarcoplasmic reticulum (SR) during diastolic depolarization in sinoatrial nodal cells (SANC) occur even in the basal state and activate an inward Na(+)-Ca2+ exchanger current that affects spontaneous beating. Why SANC can generate spontaneous LCRs under basal conditions, whereas ventricular cells cannot, has not previously been explained. Here we show that a high basal cAMP level of isolated rabbit SANC and its attendant increase in protein kinase A (PKA)-dependent phosphorylation are obligatory for the occurrence of spontaneous, basal LCRs and for spontaneous beating. Gradations in basal PKA activity, indexed by gradations in phospholamban phosphorylation effected by a specific PKA inhibitory peptide were highly correlated with concomitant gradations in LCR spatiotemporal synchronization and phase, as well as beating rate. Higher levels of basal PKA inhibition abolish LCRs and spontaneous beating ceases. Stimulation of beta-adrenergic receptors extends the range of PKA-dependent control of LCRs and beating rate beyond that in the basal state. The link between SR Ca2+ cycling and beating rate is also present in vivo, as the regulation of beating rate by local beta-adrenergic receptor stimulation of the sinoatrial node in intact dogs is markedly blunted when SR Ca2+ cycling is disrupted by ryanodine. Thus, PKA-dependent phosphorylation of proteins that regulate cell Ca2+ balance and spontaneous SR Ca2+ cycling, ie, phospholamban and L-type Ca2+ channels (and likely others not measured in this study), controls the phase and size of LCRs and the resultant Na(+)-Ca2+ exchanger current and is crucial for both basal and reserve cardiac pacemaker function.

The error-related negativity (ERN/Ne) and error positivity (Pe) have been associated with error detection and response monitoring. More recently, heart rate (HR) and skin conductance (SC) have also been shown to be sensitive to the internal detection of errors. An enhanced ERN has consistently been observed in anxious subjects and there is some suggestion that the ERN is related to general negative affective experience (NA). The ERN has been source localized to the anterior cingulate cortex-a structure implicated in the regulation of affective, response selection, and autonomic resources. Thus, the findings that autonomic measures and affective distress are related to response monitoring are consistent with anterior cingulate cortex function. In the present experiment, we sought to evaluate more comprehensively the relationship between self-reported negative affect and error-related physiology in a between-groups design. Results indicate that high NA was associated with significantly greater ERN and error-related SCR, and smaller Pe. These results are discussed in terms of anterior cingulate cortex function, psychopathology, and response monitoring.

Neurons in the brainstem implicated in the initiation of locomotion include glutamatergic, noradrenergic (NA), dopaminergic (DA), and serotonergic (5-HT) neurons giving rise to descending tracts. Glutamate antagonists block mesencephalic locomotor region-induced and spontaneous locomotion, and glutamatergic agonists induce locomotion in spinal animals. NA and 5-HT inputs to the spinal cord originate in the brainstem, while the descending dopaminergic pathway originates in the hypothalamus. Agonists acting at NA, DA or 5-HT receptors facilitate or induce locomotion in spinal animals. 5-HT neurons located in the parapyramidal region (PPR) produce locomotion when stimulated in the isolated neonatal rat brainstem-spinal cord preparation, and they constitute the first anatomically discrete group of spinally-projecting neurons demonstrated to be involved in the initiation of locomotion in mammals. Neurons in the PPR are activated during treadmill locomotion in adult rats. Locomotion evoked from the PPR is mediated by 5-HT(7) and 5-HT(2A) receptors, and 5-HT(7) antagonists block locomotion in cat, rat and mouse preparations, but have little effect in mice lacking 5-HT(7) receptors. 5-HT induced activity in 5-HT(7) knockout mice is rhythmic, but coordination among flexor and extensor motor nuclei and left and right sides of the spinal cord is disrupted. In the adult wild-type mouse, 5-HT(7) receptor antagonists impair locomotion, producing patterns of activity resembling those induced by 5-HT in 5-HT(7) knockout mice. 5-HT(7) receptor antagonists have a reduced effect on locomotion in adult 5-HT(7) receptor knockout mice. We conclude that the PPR is the source of a descending 5-HT command pathway that activates the CPG via 5-HT(7) and 5-HT(2A) receptors. Further experiments are necessary to define the putative glutamatergic, DA, and NA command pathways.

Studies using emotionally salient stimuli have demonstrated neural activation in limbic and paralimbic brain regions. In some studies, subjects passively perceive evocative stimuli, while in other studies, they perform specific cognitive tasks. Evidence is emerging that even a simple cognitive task performed on emotionally salient stimuli can affect neural activation in emotion-associated brain regions. We tested the hypothesis that rating the subjective experience of an aversive visual stimulus would decrease limbic/paralimbic activation and increase activity in medial frontal regions. Ten healthy subjects underwent (15)O PET scans while they viewed pictures of aversive (AV) and nonaversive (NA) content, taken from the International Affective Picture System. Subjects appraised pictures on a scale of pleasantness/unpleasantness during one set of scans (RTNG), and they passively viewed pictures during another set (PSVW). After each scan, emotional responses were assessed. RTNG was associated with significantly less intensity of sadness and significantly less activation (AV - NA) of the right insula/amygdala and left insula, relative to PSVW. RTNG also activated the dorsal medial prefrontal cortex and the anterior cingulate sulcus, which were not differentially activated during PSVW. For both RTNG and PSVW, subjects activated the left fusiform gyrus. The results support the proposition that task instructions about how subjects should process evocative stimuli can affect neural activity.

1. A detailed compartmental model of a cerebellar Purkinje cell with active dendritic membrane was constructed. The model was based on anatomic reconstructions of single Purkinje cells and included 10 different types of voltage-dependent channels described by Hodgkin-Huxley equations, derived from Purkinje cell-specific voltage-clamp data where available. These channels included a fast and persistent Na+ channel, three voltage-dependent K+ channels, T-type and P-type Ca2+ channels, and two types of Ca(2+)-activated K+ channels. 2. The ionic channels were distributed differentially over three zones of the model, with Na+ channels in the soma, fast K+ channels in the soma and main dendrite, and Ca2+ channels and Ca(2+)-activated K+ channels in the entire dendrite. Channel densities in the model were varied until it could reproduce Purkinje cell responses to current injections in the soma or dendrite, as observed in slice recordings. 3. As in real Purkinje cells, the model generated two types of spiking behavior. In response to small current injections the model fired exclusively fast somatic spikes. These somatic spikes were caused by Na+ channels and repolarized by the delayed rectifier. When higher-amplitude current injections were given, sodium spiking increased in frequency until the model generated large dendritic Ca2+ spikes. Analysis of membrane currents underlying this behavior showed that these Ca2+ spikes were caused by the P-type Ca2+ channel and repolarized by the BK-type Ca(2+)-activated K+ channel. As in pharmacological blocking experiments, removal of Na+ channels abolished the fast spikes and removal of Ca2+ channels removed Ca2+ spiking. 4. In addition to spiking behavior, the model also produced slow plateau potentials in both the dendrite and soma. These longer-duration potentials occurred in response to both short and prolonged current steps. Analysis of the model demonstrated that the plateau potentials in the soma were caused by the window current component of the fast Na+ current, which was much larger than the current through the persistent Na+ channels. Plateau potentials in the dendrite were carried by the same P-type Ca2+ channel that was also responsible for Ca2+ spike generation. The P channel could participate in both model functions because of the low-threshold K2-type Ca(2+)-activated K+ channel, which dynamically changed the threshold for dendritic spike generation through a negative feedback loop with the activation kinetics of the P-type Ca2+ channel. 5. These model responses were robust to changes in the densities of all of the ionic channels.(ABSTRACT TRUNCATED AT 400 WORDS)

In previous papers [Harding (2001), Acta Cryst. D57, 401-411, and references therein] the geometry of metal-ligand interactions was examined for six metals (Ca, Mg, Mn, Fe, Cu, Zn) using the Protein Data Bank and compared with information from accurately determined structures of relevant small-molecule crystals in the Cambridge Structural Database. Here, the environments of Na(+) and K(+) ions found in protein crystal structures are examined in an equivalent way. Target M(+).O distances are proposed and the agreement with observed distances is summarized. The commonest interactions are with water molecules and the next commonest with main-chain carbonyl O atoms.

1. Electrophysiological techniques are described which allow intracellular recording from peripheral myelinated axons of lizards and frogs for up to several hours. The sciatic and intramuscular axons studied here have resting potentials of -60 to -80 mV and action potentials (evoked by stimulation of the proximal nerve trunk) of 50-90 mV. They show a prominent depolarizing afterpotential (d.a.p.), which is present both in isolated axons and in axons still attached to their peripheral terminals. This d.a.p. has a peak amplitude of 5-20 mV at the resting potential, and decays with a half-time of 20-100 msec.2. The peak amplitude of the d.a.p. is voltage-sensitive, increasing to up to 26 mV with membrane hyperpolarization. The d.a.p. disappears as the axon is depolarized to -60 to -45 mV, and does not appear to reverse with further depolarization.3. The d.a.p. is not reduced when bath Ca is replaced by 2-10 mm divalent Mn or Ni. The d.a.p. is not reversed when axons depleted of Cl (by prolonged exposure to Cl-deficient, SO(4)-enriched solutions) are bathed in Cl-rich solutions. These results suggest that the d.a.p. is not mediated by a conductance change specific for Ca or Cl ions. Partial substitution of tetramethylammonium for bath Na, or addition of 10(-5)m-tetrodotoxin to the normal bathing solution, reduces the amplitude of both the action potential and the d.a.p.4. The amplitude of the d.a.p. is not sensitive to bath [K] over the range 1-7.5 mm, provided that all measurements are made at the same holding potential. This result argues that the d.a.p. is not mediated by accumulation of K outside the active axon.5. Treatments expected to inhibit the Na-K exchange pump (cooling from 25 to 10 degrees C, or 0.15 mm-ouabain) do not enlarge or prolong the d.a.p., although they do abolish a slower hyperpolarizing afterpotential seen following repetitive stimulation.6. The passive voltage response of the axon to small injected pulses of depolarizing or hyperpolarizing current shows a prominent, slowly decaying component with a time course similar to that of the d.a.p. Depolarizing current reduces the input resistance of the axon, and increases the rate of decay of both the passive voltage response and the d.a.p. There is a slight conductance increase during the peak of the d.a.p., but the same conductance increase can be produced by a comparable passive depolarization.7. We conclude that the d.a.p. is due mainly to a passive capacitative current, probably resulting from discharge of the internodal axonal membrane capacitance through a resistive current pathway beneath or through the myelin sheath. We suggest that this slow capacitative discharge becomes evident as soon as most of the nodal ionic channels activated during the action potential have closed. An electrical model of the myelinated axon that incorporates the postulated internodal leakage pathway can account both for the prolonged d.a.p. recorded inside the axon, and for the potential profile recorded extra-axonally in or near the internodal periaxonal space.

BACKGROUND

Although we know much about the molecular makeup of the sinus node (SN) in small mammals, little is known about it in humans. The aims of the present study were to investigate the expression of ion channels in the human SN and to use the data to predict electrical activity.

RESULTS

Quantitative polymerase chain reaction, in situ hybridization, and immunofluorescence were used to analyze 6 human tissue samples. Messenger RNA (mRNA) for 120 ion channels (and some related proteins) was measured in the SN, a novel paranodal area, and the right atrium (RA). The results showed, for example, that in the SN compared with the RA, there was a lower expression of Na(v)1.5, K(v)4.3, K(v)1.5, ERG, K(ir)2.1, K(ir)6.2, RyR2, SERCA2a, Cx40, and Cx43 mRNAs but a higher expression of Ca(v)1.3, Ca(v)3.1, HCN1, and HCN4 mRNAs. The expression pattern of many ion channels in the paranodal area was intermediate between that of the SN and RA; however, compared with the SN and RA, the paranodal area showed greater expression of K(v)4.2, K(ir)6.1, TASK1, SK2, and MiRP2. Expression of ion channel proteins was in agreement with expression of the corresponding mRNAs. The levels of mRNA in the SN, as a percentage of those in the RA, were used to estimate conductances of key ionic currents as a percentage of those in a mathematical model of human atrial action potential. The resulting SN model successfully produced pacemaking.

CONCLUSIONS

Ion channels show a complex and heterogeneous pattern of expression in the SN, paranodal area, and RA in humans, and the expression pattern is appropriate to explain pacemaking.

Tenofovir disoproxil fumarate (TDF) has demonstrated high antiviral efficacy in treatment-naive patients with chronic hepatitis B virus (HBV) infection but experience in nucleoside/nucleotide analogue (NA)-experienced patients is limited. In this retrospective multicenter study we therefore assessed the long-term efficacy of TDF monotherapy in patients with prior failure or resistance to different NA treatments. Criteria for inclusion were HBV DNA levels >4.0 log(10) copies/mL at the start and a minimum period of TDF therapy for at least 6 months. In all, 131 patients (mean age 42 +/- 12 years, 95 male, 65% hepatitis B e antigen [HBeAg]-positive) were eligible. Pretreatment consisted of either monotherapy with lamivudine (LAM; n = 18), adefovir (ADV; n = 8), and sequential LAM-ADV therapy (n = 73), or add-on combination therapy with both drugs (n = 29). Three patients had failed entecavir therapy. Resistance analysis in 113 of the 131 patients revealed genotypic LAM and ADV resistance in 62% and 19% of patients, respectively. The mean HBV DNA level at TDF baseline was 7.6 +/- 1.5 log(10) copies/mL. The overall cumulative proportion of patients achieving HBV DNA levels <400 copies/mL was 79% after a mean treatment duration of 23 months (range, 6-60). Although LAM resistance did not influence the antiviral efficacy of TDF, the presence of ADV resistance impaired TDF efficacy (100% versus 52% probability of HBV DNA <400 copies/mL, respectively). However, virologic breakthrough was not observed in any of the patients during the entire observation period. Loss of HBeAg occurred in 24% of patients and HBsAg loss occurred in 3%. No significant adverse events were noticed during TDF monotherapy.

CONCLUSIONS

TDF monotherapy induced a potent and long-lasting antiviral response in NA-experienced patients with previous treatment failure. Our data may have implications for current add-on strategies.

The composition of the intestinal luminal content varies considerably with diet. It is important therefore that the intestinal epithelium senses and responds to these significant changes and regulates its functions accordingly. Although it is becoming evident that the gut epithelium senses and responds to luminal nutrients, little is known about the nature of the nutrient sensing molecule and the downstream cellular events. A prototype example is the modulation in the capacity of the gut to absorb monosaccharides via the intestinal luminal membrane Na(+)/glucose cotransporter, SGLT1. The experimental evidence suggests that luminal sugar is sensed by a glucose sensor residing on the luminal membrane of the gut epithelium and linked to a G-protein-coupled receptor, cAMP/PKA (protein kinase A) pathway, resulting ultimately in modulation of intestinal monosaccharide absorption. Here we report the expression, at mRNA and protein levels, of members of the T1R sweet taste receptors, and the alpha-subunit of the G-protein gustducin, in the small intestine and the enteroendocrine cell line, STC-1. In the small intestine, there is a highly coordinated expression of sweet taste receptors and gustducin, a G-protein implicated in intracellular taste signal transduction, throughout the gut. The potential involvement of these receptors in sugar sensing in the intestine will facilitate our understanding of intestinal nutrient sensing, with implications for better nutrition and health maintenance.

ADP-ribosyl cyclase catalyzes the cyclization of NAD+ to produce cyclic ADP-ribose (cADPR), which is emerging as an endogenous regulator of the Ca(2+)-induced Ca2+ release mechanism in cells. CD38 is a lymphocyte differentiation antigen which has recently been shown to be a bifunctional enzyme that can synthesize cADPR from NAD+ as well as hydrolyze cADPR to ADP-ribose. In this study, we show that both the cyclase and CD38 can also catalyze the exchange of the nicotinamide group of NADP+ with nicotine acid (NA). The product is nicotinic acid adenine dinucleotide phosphate (NAADP+), a metabolite we have previously shown to be potent in Ca2+ mobilization (Lee, H. C., and Aarhus, R. (1995) J. Biol. Chem. 270, 2152-2157). The switch of the catalysis to the exchange reaction requires acidic pH and NA. The half-maximal effective concentration of NA is about 5 mM for both the cyclase and CD38. In the absence of NA or at neutral pH, the cyclase converts NADP+ to another metabolite, which is identified as cyclic ADP-ribose 2'-phosphate. Under the same conditions, CD38 converts NADP+ to ADP-ribose 2'-phosphate instead, which is the hydrolysis product of cyclic ADP-ribose 2'-phosphate. That two different products of ADP-ribosyl cyclase and CD38, cADPR and NAADP+, are both involved in Ca2+ mobilization suggests a crucial role of these enzymes in Ca2+ signaling.

Tetrodotoxin (TTX)-sensitive Na currents were examined in single dissociated ventricular myocytes from neonatal rats. Single channel and whole cell currents were measured using the patch-clamp method. The channel density was calculated as 2/micron 2, which agreed with our usual finding of four channels per membrane patch. At 20 degrees C, the single channel conductance was 20 pS. The open time distributions were fit by a single-exponential function with a mean open time of approximately 1.0 ms at membrane potentials from -60 to -40 mV. Averaged single channel and whole cell currents were similar when scaled and showed both fast and slow rates of inactivation. The inactivation and activation gating shifted quickly to hyperpolarized potentials for channels in cell-attached as well as excised patches, whereas a much slower shift occurred in whole cells. Slowly inactivating currents were present in both whole cell and single channel current measurements at potentials as positive as -40 mV. In whole cell measurements, the potential range could be extended, and slow inactivation was present at potentials as positive as -10 mV. The curves relating steady state activation and inactivation to membrane potential had very little overlap, and slow inactivation occurred at potentials that were positive to the overlap. Slow inactivation is in this way distinguishable from the overlap or window current, and the slowly inactivating current may contribute to the plateau of the rat cardiac action potential. On rare occasions, a second set of Na channels having a smaller unit conductance and briefer duration was observed. However, a separate set of threshold channels, as described by Gilly and Armstrong (1984. Nature [Lond.]. 309:448), was not found. For the commonly observed Na channels, the number of openings in some samples far exceeded the number of channels per patch and the latencies to first opening or waiting times were not sufficiently dispersed to account for the slowly inactivating currents: the slow inactivation was produced by channel reopening. A general model was developed to predict the number of openings in each sample. Models in which the number of openings per sample was due to a dispersion of waiting times combined with a rapid transition from an open to an absorbing inactivated state were unsatisfactory and a model that was more consistent with the results was identified.

Little is known about the physiology of neurons in Caenorhabditis elegans. Using new techniques for in situ patch-clamp recording in C. elegans, we analyzed the electrical properties of an identified sensory neuron (ASER) across four developmental stages and 42 unidentified neurons at one stage. We find that ASER is nearly isopotential and fails to generate classical Na+ action potentials. Rather, ASER displays a high sensitivity to input currents coupled to a depolarization-dependent reduction in sensitivity that may endow ASER with a wide dynamic range. Voltage clamp revealed depolarization-activated K+ and Ca2+ currents that contribute to high sensitivity near the zero-current potential. The depolarization-dependent reduction in sensitivity can be attributed to activation of K+ current at voltages where it dominates the net membrane current. The voltage dependence of membrane current was similar in all neurons examined, suggesting that C. elegans neurons share a common mechanism of sensitivity and dynamic range.

Using kinetic data from three different K+ currents in acutely isolated neurons, a single electrical compartment representing the soma of a ventral cochlear nucleus (VCN) neuron was created. The K+ currents include a fast transient current (IA), a slow-inactivating low-threshold current (ILT), and a noninactivating high-threshold current (IHT). The model also includes a fast-inactivating Na+ current, a hyperpolarization-activated cation current (Ih), and 1-50 auditory nerve synapses. With this model, the role IA, ILT, and IHT play in shaping the discharge patterns of VCN cells is explored. Simulation results indicate that IHT mainly functions to repolarize the membrane during an action potential, and IA functions to modulate the rate of repetitive firing. ILT is found to be responsible for the phasic discharge pattern observed in Type II cells (bushy cells). However, by adjusting the strength of ILT, both phasic and regular discharge patterns are observed, demonstrating that a critical level of ILT is necessary to produce the Type II response. Simulated Type II cells have a significantly faster membrane time constant in comparison to Type I cells (stellate cells) and are therefore better suited to preserve temporal information in their auditory nerve inputs by acting as precise coincidence detectors and having a short refractory period. Finally, we demonstrate that modulation of Ih, which changes the resting membrane potential, is a more effective means of modulating the activation level of ILT than simply modulating ILT itself. This result may explain why ILT and Ih are often coexpressed throughout the nervous system.

The response of retinal rod photoreceptors to light consists of a membrane hyperpolarization resulting from the decrease of a light-sensitive conductance in the outer segment. According to the calcium hypothesis, this conductance is blocked by a rise in intracellular free Ca triggered by light, a notion supported by the findings that an induced rise in internal Ca leads to blockage of the light-sensitive conductance and that light triggers a net Ca efflux from the outer segment via a Na-Ca exchanger, suggesting a rise in internal free Ca in the light. We have now measured both Ca influx and efflux through the outer segment plasma membrane and find that, contrary to the calcium hypothesis, light seems to decrease rather than increase the free Ca concentration in the rod outer segment. This result implies that Ca does not mediate visual excitation but it probably has a role in light adaptation.

Polymorphisms in the gene encoding sterile 20/SPS1-related proline/alanine-rich kinase (SPAK) associate with hypertension susceptibility in humans. SPAK interacts with WNK kinases to regulate the Na(+)-K(+)-2Cl(-) and Na(+)-Cl(-) co-transporters [collectively, N(K)CC]. Mutations in WNK1/4 and N(K)CC can cause changes in BP and dyskalemia in humans, but the physiologic role of SPAK in vivo is unknown. We generated and analyzed SPAK-null mice by targeting disruption of exons 9 and 10 of SPAK. Compared with SPAK(+/+) littermates, SPAK(+/-) mice exhibited hypotension without significant electrolyte abnormalities, and SPAK(-/-) mice not only exhibited hypotension but also recapitulated Gitelman syndrome with hypokalemia, hypomagnesemia, and hypocalciuria. In the kidney tissues of SPAK(-/-) mice, the expression of total and phosphorylated (p-)NCC was markedly decreased, but that of p-OSR1, total NKCC2, and p-NKCC2 was significantly increased. We observed a blunted response to thiazide but normal response to furosemide in SPAK(-/-) mice. In aortic tissues, total NKCC1 expression was increased but p-NKCC1 was decreased in SPAK-deficient mice. Both SPAK(+/-) and SPAK(-/-) mice had impaired responses to the selective α(1)-adrenergic agonist phenylephrine and the NKCC1 inhibitor bumetanide, suggesting that impaired aortic contractility may contribute to the hypotension of SPAK-null mice. In summary, SPAK-null mice have defects of NCC in the kidneys and NKCC1 in the blood vessels, leading to hypotension through renal salt wasting and vasodilation. SPAK may be a promising target for antihypertensive therapy.

1. A method for producing rapid [Ca2+] and [Sr2+] changes in the frog skinned muscle fibre preparation while maintaining constant all other cationic concentrations (Moisescu, 1976a, b) is described and analysed in detail. 2. Different experiments, some of them involving the Ca2+-sensitive photoprotein aequorin, as well as theoretical considerations, indicate that with this method one can produce a Ca2+ (or Sr2+) concentration change within 0.1--0.15 sec in a whole preparation having a diameter of 50 micrometer. 3. The rate of force development was similar to that observed in vivo. 4. The radial diffusion coefficient of EGTA in relaxed myofibrillar preparations was measured and found to be 4.6 x 10(-6) cm2sec-1 at 20 degrees C. 5. The sarcoplasmic reticulum in myofibrillar bundles was found to be active with respect to both Ca2+ and Sr2+ in the solutions used ([Mg2+] 1 mM; [Na] 30 mM; [K] 140-170 mM; [Cl] less than or equal to 20 mM; pH 7.10). 6. The amount of Ca released by caffeine from internal stores (previously loaded with Ca) can raise the total Ca concentration in the muscle fibre preparation by at least 1.8 mM. 7. The presence of 10 mM-caffeine in all bathing solutions reduced drastically the ability of the sarcoplasmic reticulum to accumulate both Ca and Sr.

1. Properties of the pace-maker current (if) in Purkinje fibres were studied in the presence of Ba, which by partially blocking the iK1 channel reduces K depletion during hyperpolarizing voltage-clamp pulses, and eliminates the main cause of distortion in the current time course. 2. On raising the external potassium concentration (Kb), the if fully activated current--voltage relation (if(E)) increases in the inward direction. In the range 3--36 mM--Kb and negative to -50 mV the current is inward, and no cross-over is observed. 3. In normal conditions, the reversal potential (Ef) for if lies in the voltage region positive to -50 mV, and can be observed on lowering the external sodium concentration (Nab). Ef shifts to the negative direction when Nab is decreased. Slopes ranging between 29 and 35 mV/decade are found for Nernst plots of Ef against Nab. Changing Nab in the range 140--4.4 mM causes the if(E) relation to undergo a simple shift along the voltage axis, without significant change in its slope. 4. Ef also depends on Kb, as can be observed in low Nab (35 mM), and shifts to the positive direction by about 26 mV for every 10-fold change in Kb. The fully activated slope conductance increases when Kb is increased. 5. It is concluded that Na and K both participate in carrying if. The slope of the fully activated if(E) relation increases with Kb, but is unchanged in different Nab, indicating that the channel conductance depends on Kb, but not appreciably on Nab.

1. 45-Ca uptake by pinched-off nerve terminals (synaptosomes) of rat brain incubated in standard physiological saline (including 132 mM-Na + 5mM-K + 1-2 mM-Ca) at 30 degrees C averages about 0-5 mumole Ca per g protein per minute. This may be equivalent to a Ca influx of about 0-03 p-mole/cm-2 sec. 2. The rate of 45-Ca uptake is increased when the concentration of K in the medium is increased above 15-20 mM, K replacing Na isosmotically. Maximum stimulation, a three- to six-fold increase in the rate of Ca uptake, occurs when [K]o is about 60 mM. The effect of increased [K]o is reversible. 3. The K-stimulated Ca uptake is associated primarily with the nerve terminal fraction of brain homogenates. The entering Ca is not accompanied by extracellular markers such as mannitol or inulin. Replacement of external chloride by methylsulphate or sulphate does not prevent the stimulation by K. 4. The effects of external K are quantitatively mimicked by Rb. Caesium also stimulates Ca uptake, but is only about one fifth as effective as K or Rb; Li is ineffective. 5. Two other depolarizing agents also stimulate Ca uptake by synaptosomes: veratridine (7-5 times 10- minus 6 to 7-5 times 10- minus 5 M) and scorpion (Leirus quinquestriatus) venom (6-7 times 10- minus 7 to 6-7 times 10- minus g/ml.). The stimulatory effects of veratridine and scorpion venom, but not of increased [K] are blocked by 2 times 10- minus 7 M tetrodotoxin. 6. Internal K also influences the rate of 45-Ca uptake by synaptosomes: lowering [K]i reduces the stimulatory effect of external K and veratridine. 7. Replacement of external Na by choline markedly inhibits the response to veratridine, but has a much smaller effect on the response to increased [K]o. 8. The Ca uptake mechanism has an apparent dissociation constant for Ca (KCa) of about 0-8 mM. Increasing [K]o increases the maximal rate of Ca uptake, but has no effect on KCa. The K-induced 45-Ca uptake is competitively inhibited by Mg-2+, Mn-2+ and La-3+. 9. The release of acetylcholine and noradrenaline was also studied. Increasing [K]o stimulates external Ca-dependent acetylcholine release. Scorpion venom stimulates noradrenaline release from synaptosomes; this effect could be prevented by adding tetrodotoxin or removing external Ca. 10. These results indicate that synaptosomes may increase their permeability to Ca, accumulate Ca and release neural transmitter substances, when stimulated by depolarizing agents under appropriate physiological conditions.

Forebrain serotonergic lesions attenuate the ability of d-amphetamine to decrease impulsivity in a delay-discounting paradigm, potentially through interactions between the serotonin (5-HT) and dopamine (DA) systems. Nucleus accumbens (NAC) lesions increase impulsivity, but the extent to which accumbal DA is involved in regulating impulsive choice is unknown. In the current study, the effects of intra-accumbal infusions of 6-hydroxydopamine (6-OHDA) on impulsive choice were evaluated, in combination with d-amphetamine and serotonergic drugs, in order to investigate the importance of 5-HT : DA interactions in the control of impulsive behavior. Following training on a delay-discounting task, animals received intra-NAC 6-OHDA or sham surgery. Postoperatively, subjects received systemic injections of d-amphetamine (0, 0.3, 1.0, 1.5 mg/kg) and the 5-HT(1A) receptor agonist 8-OH-DPAT (0, 0.1, 0.3, 1.0 mg/kg). Intra-NAC 6-OHDA, which reduced local DA and NA levels by 70-75%, had no effect on delay-discounting, but transiently potentiated the d-amphetamine-induced decrease in impulsive choice. 8-OH-DPAT (1.0 mg/kg) increased impulsivity in sham-operated controls, an effect which was blocked by the 5-HT(1A) receptor antagonist WAY 100635. However, 8-OH-DPAT had no effect on impulsivity in 6-OHDA NAC lesioned rats. 8-OH-DPAT (0.3 mg/kg), which did not itself alter task performance, blocked the effect of d-amphetamine in sham-operated controls, while WAY 100635 augmented the effect of amphetamine in all subjects. In an additional experiment, intracerebroventricular administration of the selective serotonergic toxin 5,7-dihydroxytryptamine, which decreased forebrain 5-HT levels by 85-90%, did not block 8-OH-DPAT's ability to increase impulsive choice. These data suggest a significant role for 5-HT : DA interactions within the NAC in the control of impulsivity, and in the mechanism by which amphetamine decreases impulsive choice.