In humans, subjects homozygous for arginine (ArgArg) at codon 16 of the beta2-adrenergic receptor (beta2AR) have been shown to have greater agonist-mediated desensitization than subjects homozygous for glycine (GlyGly). We sought to determine if this substitution differentially influenced cardiovascular function during short duration (9 min) low and high intensity exercise (40 and 75% of peak work). Healthy Caucasian ArgArg (n = 16), GlyGly (n = 31) and ArgGly (n = 17) subjects matched for age, sex and peak oxygen uptake were studied. There were no differences in adrenaline (ADR) at rest or with heavy exercise, but the ArgArg group had lower ADR with light exercise (P = 0.04). Resting heart rate (HR) was higher in ArgArg (P < 0.01), while cardiac output (Q), stroke volume (SV), and mean arterial pressure (MAP) were lower than the other groups (HR = 86+/-2, 78+/-2, 80+/-1 beats min(-1); Q = 5.7+/-0.81, 6.1+/-0.18, 6.7+/-0.22 l min(-1); SV = 68+/-3, 82+/-3, 89+/-4 ml beat(-1); MAP = 92+/-1, 103+/-2, 98+/-1 mmHg-- for ArgArg, ArgGly and GlyGly, respectively, means +/-s.e.m., P < 0.01), however, no differences were observed in systemic vascular resistance (SVR). With low intensity exercise and high intensity exercise the ArgArg group continued to have a lower , SV and MAP compared to the other groups (P < 0.05), with no differences observed in SVR. During recovery, the ArgArg subjects continued to have a lower MAP but there were no differences in HR, , or SVR. These data suggest that subjects homozygous for Arg at codon 16 of the beta2AR have reduced and MAP at rest that persist during exercise with no evidence for differential changes over the course of exercise despite large changes in catecholamines. This may suggest possible genotype-related differences in baseline receptor function or density which causes phenotypic differences at rest that are sustained during short-term exercise.

OBJECTIVE

The aim of this study was to investigate associations between circulating catecholamines, endothelial damage, and coagulopathy in experimental human endotoxemia and septic patients.

METHODS

Nine healthy male volunteers undergoing endotoxemia (4-hour 0.5 ng/kg/hour infusion of E. coli lipopolysaccharide, blood sampling at 0, 4, and 6 hours) and 20 patients with severe sepsis. Analysis of plasma biomarkers (adrenaline, noradrenaline, thrombomodulin, syndecan-1, soluble vascular endothelial cadherin, histone-complexed DNA fragments, soluble CD40 ligand [sCD40L], protein C, tissue-type plasminogen activator, plasminogen activator inhibitor 1) and routine coagulation tests.

RESULTS

Endotoxemia increased heart rate, temperature, white blood cell count, C-reactive protein and procalcitonin, decreased blood pressure and induced a hemostatic response with platelet consumption, reduced protein C and sCD40L levels and enhanced tissue-type plasminogen activator release (all P<.05). Septic patients had increased levels of noradrenaline, syndecan-1, thrombomodulin, histone-complexed DNA and sCD40L but reduced soluble vascular endothelial cadherin and plasminogen activator inhibitor 1 (all P<.05) and plasma catecholamines correlated positively with syndecan-1 (adrenaline and noradrenaline) and sTM (only noradrenaline) (all P<.05), biomarkers reflecting endothelial damage. Furthermore, noradrenaline, syndecan-1 and thrombomodulin levels correlated with INR and disease severity scores (noradrenaline and thrombomodulin) (all P<.05).

CONCLUSIONS

Experimental endotoxemia induced a discrete hemostatic response without sympathoadrenal activation or endothelial damage. Septic patients had high levels of catecholamines and endothelial damage biomarkers that correlated with each other and with markers of hypocoagulability and disease severity.

The alpha2A- and alpha2C-adrenoceptor subtypes were evaluated in postmortem brains from suicides with depression (n = 22), suicides with other diagnoses (n = 12), and controls (n = 26). Membrane assays with the antagonist [3H]RX821002 (2-[3H]methoxyidazoxan) suggested the presence of alpha2A-adrenoceptors in the frontal cortex and both alpha2C-adrenoceptors and alpha2A-adrenoceptors in the caudate. The proportions in caudate were similar in controls (alpha2A, 86%; alpha2C, 14%), depressed suicides (alpha2A, 91%; alpha2C, 9%), and suicides with other diagnoses (alpha2A, 88%; alpha2C, 12%). Autoradiography of [3H]RX821002 binding under alpha(2B/C)-adrenoceptor-masking conditions confirmed the similar densities of alpha2A-adrenoceptors in the cortex, hippocampus, and striatum from controls and suicides. In the frontal cortex of depressed suicides, competition of [3H]RX821002 binding by (-)-adrenaline revealed a greater proportion (61 +/- 9%) of alpha2A-adrenoceptors in the high-affinity conformation for agonists than in controls (39 +/- 5%). Simultaneous analysis with the agonists [3H]clonidine and [3H]UK14304 and the antagonist [3H]RX821002 in the same depressed suicides confirmed the enhanced alpha2A-adrenoceptor density when evaluated by agonist, but not by antagonist, radioligands. The results indicate that depression is associated with a selective increase in the high-affinity conformation of the brain alpha2A-adrenoceptors.

BACKGROUND

Current clinical advice regarding peanut allergy is based on small series of patients.

OBJECTIVE

To determine, in a large group of peanut allergic subjects, the patterns of clinical severity, symptom progression and availability and use of rescue medications.

METHODS

Questionnaire study of 622 self-reported allergic subjects.

RESULTS

A total of 406 patients (66%) reported symptoms on contact with peanut. Only 121 (19%) had been knowingly exposed to peanut before the first documented reaction, implying a high frequency of occult sensitization. Severe symptoms were more common in adults. Abdominal symptoms were significantly associated with collapse. Fifty per cent reported reactions in the previous year. Only 82 (13%) had been admitted to hospital because of a reaction. Adrenaline was carried in some form by 65% though only 78 subjects (12.5%) had ever received injected adrenaline. Only 18/43 subjects (41%) who collapsed were given adrenaline. Skin-prick test weal size correlated weakly with severity but there were large overlaps between the groups. Peanut-specific IgE peaked in the teenage group, but did not correlate with severity.

CONCLUSIONS

Peanut allergy is characterized by more severe symptoms than other food allergies and by high rates of symptoms on minimal contact. Skin-prick testing and peanut-specific IgE levels do not predict clinical severity. Avoidance of peanut is difficult. Many people suffering severe relations are inadequately treated. Sufferers need education and training in the use of rescue medication.

The family of adrenergic receptors contains nine different subtypes of G protein-coupled receptors which mediate the biological effects of adrenaline and noradrenaline. With few exceptions, the full therapeutic potential of subtype-selective therapy has not yet been explored for the group of adrenergic receptors. In the absence of sufficiently subtype-selective ligands which can distinguish between individual receptor subtypes of the adrenergic family, gene-targeted mouse models with deletions in these receptor genes have recently been generated and characterized. These genetic mouse models have helped to assign specific pharmacological effects of alpha(2)-receptor agonists or antagonists to individual receptor subtypes. However, some unexpected and novel functions of alpha(2)-adrenergic receptors were also uncovered in these mouse models: Presynaptic control of catecholamine release from adrenergic nerves in the central and sympathetic nervous system may be regulated by three different alpha(2)-receptor subtypes, alpha(2A), alpha(2B), and alpha(2C). A similar feedback loop also controls the release of catecholamines from the adrenal gland. alpha(2B)-receptors are not only involved in regulating vascular tone in the adult organism, but they are essential for the development of the vascular system of the placenta during prenatal development. The challenge will now be to generate strategies to identify whether the findings obtained in gene-targeted mice may predict the action of receptor subtype-selective drugs in humans.

The brain is a vital organ, susceptible to alterations under genetic influences and environmental experiences. Social isolation (SI) acts as a stressor which results in alterations in reactivity to stress, social behavior, function of neurochemical and neuroendocrine system, physiological, anatomical and behavioral changes in both animal and humans. During early stages of life, acute or chronic SIS has been proposed to show signs and symptoms of psychiatric and neurological disorders such as anxiety, depression, schizophrenia, epilepsy and memory loss. Exposure to social isolation stress induces a variety of endocrinological changes including the activation of hypothalamic-pituitary-adrenal (HPA) axis, culminating in the release of glucocorticoids (GCs), release of catecholamines, activation of the sympatho-adrenomedullary system, release of Oxytocin and vasopressin. In several regions of the central nervous system (CNS), SIS alters the level of neurotransmitter such as dopamine, serotonin, gamma aminobutyric acid (GABA), glutamate, nitrergic system and adrenaline as well as leads to alteration in receptor sensitivity of N-methyl-D-aspartate (NMDA) and opioid system. A change in the function of oxidative and nitrosative stress (O&NS) mediated mitochondrial dysfunction, inflammatory factors, neurotrophins and neurotrophicfactors (NTFs), early growth response transcription factor genes (Egr) and C-Fos expression are also involved as a pathophysiological consequences of SIS which induce neurological and psychiatric disorders.

Glucose is an essential metabolic substrate for all bodily tissues. The brain depends particularly on a constant supply of glucose to satisfy its energy demands. Fortunately, a complex physiological system has evolved to keep blood glucose at a constant level. The consequences of poor glucose homeostasis are well-known: hyperglycemia associated with uncontrolled diabetes can lead to cardiovascular disease, neuropathy and nephropathy, while hypoglycemia can lead to convulsions, loss of consciousness, coma, and even death. The glucose counterregulatory response involves detection of declining plasma glucose levels and secretion of several hormones including glucagon, adrenaline, cortisol, and growth hormone (GH) to orchestrate the recovery from hypoglycemia. Low blood glucose leads to a low brain glucose level that is detected by glucose-sensing neurons located in several brain regions such as the ventromedial hypothalamus, the perifornical region of the lateral hypothalamus, the arcuate nucleus (ARC), and in several hindbrain regions. This review will describe the importance of the glucose counterregulatory system and what is known of the neurocircuitry that underpins it.

The use of norepinephrine, and probably vasopressor therapy in general, in intensive care patients with hypotensive vasodilatation despite fluid resuscitation and evidence of acute kidney injury remains the subject of much debate and controversy. Although there is concern about the use of these drugs, these concerns are unfounded. At this time, the experimental and human data strongly suggest that, in these patients, vasopressor therapy is safe and probably beneficial from a renal, and probably general, point of view. On the basis of currently available evidence, in hypotensive vasodilated patients with acute kidney injury, restoration of blood pressure within autoregulatory values should occur promptly with noradrenaline and be sustained until such vasodilatation dissipates. The additional role of other vasopressors in these situations remains unclear. The addition of vasopressin may be helpful in individual patients, but widespread use is not supported by evidence. Alpha-dose dopamine has no advantages over noradrenaline and is not as reliably effective in restoring blood pressure and urine output. Its widespread use cannot be supported in patients with vasodilatation and acute kidney injury. Other vasopressor drugs such as epinephrine and phenylephrine may be similar in efficacy to noradrenaline. However, experience and available data with their use is vastly less than with noradrenaline. Adrenaline, in addition, is associated with hyperglycemia, hyperlactatemia, acidosis, and hypokalemia. Terlipressin appears useful in patients with acute kidney injury secondary to hepatorenal syndrome. Whether it is superior to noradrenaline in this setting remains uncertain, and more studies are needed before recommendations can be made.

1. The blocking action of adrenaline on the superior cervical ganglion of the rabbit was investigated with intracellular recording techniques.2. Adrenaline (10(-5)M) blocked initiation of post-synaptic action potentials and decreased the amplitude of excitatory post-synaptic potentials (e.p.s.p.s), but did not hyperpolarize the post-synaptic membrane.3. The depressant action of adrenaline was antagonized by phenoxy-benzamine and dihydroergotamine.4. Acetylcholine depolarizations from iontophoretic ACh were not affected by adrenaline.5. Adrenaline decreased the frequency of miniature excitatory post-synaptic potentials (m.e.p.s.p.s) and decreased the quantal content of e.p.s.p.s in a low [Ca(2+)]: [Mg(2+)] media.6. It was concluded that adrenaline blocks ganglionic transmission by acting at an alpha-adrenoceptive site in the presynaptic nerve terminals.

OBJECTIVE

Optimal monitoring of cardiac output and intravascular volume is of paramount importance for good fluid management of patients with subarachnoid hemorrhage (SAH). The aim of this study was to demonstrate the feasibility of advanced hemodynamic monitoring with transpulmonary thermodilution and to provide descriptive data early after SAH.

METHODS

Forty-six patients with SAH treated within 24 hours of the ictus were investigated. Specific targets for cardiac index >>or=3.0 L x min(-1) x m(-2)), global end-diastolic volume index (700 to 900 mL/m(2)), and extravascular lung water index (<or=14 mL/kg) were established by the single-indicator transpulmonary thermodilution technique, and a fluid management protocol emphasizing supplemental colloid administration was used to attain these targets. Plasma hormones related to stress and fluid regulation were also measured.

RESULTS

A higher cardiac index (mean value of 5.3 L x min(-1) x m(-2)) and a lower global end-diastolic volume index (555 mL/m(2)) were observed on initial measurement, for which elevations of plasma adrenaline, noradrenaline, and cortisol were also detected. Cardiac index was progressively decreased (3.5 L x min(-1) x m(-2)) and global end-diastolic volume index was normalized by fluid administration aimed at normovolemia. The extent of the initial hemodynamic and hormonal profile was greater in patients with a poor clinical status (P<0.05). The extravascular lung water index was mildly elevated but within the target range throughout the study period. No patients developed pulmonary edema or congestive heart failure.

CONCLUSIONS

The impact of sympathetic hyperactivity after SAH predisposes patients to a hyperdynamic and hypovolemic state, especially in those whose clinical status is poor. Bedside monitoring with the transpulmonary thermodilution system may be a powerful tool for the systemic management of such patients.

Eighteen poor sleepers and 18 good sleepers of mean age 52 yr, selected on the basis of their stated opinions about their sleep, were studied as pairs matched for sex, age, height and weight, on five consecutive nights and two consecutive days. Using EEG measures, the poor sleepers woke more often in the early hours of sleep and achieved half an hour less sleep. They had higher body temperatures by day and night and were more anxious. They tended to have higher urinary cortisol and adrenaline excretion. The groups did not differ in reaction time nor in excretion of urinary 3-methylhistidine. The poor sleepers over-estimated their sleep latency and both groups under-estimated their total sleep, the poor sleepers being significantly more inaccurate. It is concluded that those who complain of poor sleep have also metabolic differences from good sleepers.

Choline 2:6-xylyl ether bromide (TM 10), given systemically to cats in doses of 5 to 15 mg./kg., abolishes the effects of adrenergic nerve stimulation whilst leaving the reactions of the effector organs to adrenaline unimpaired. The effects of a single dose may take up to one hour to become fully established and last for more than twenty-four hours. Apart from transitory ganglionic blockade, cholinergic autonomic nerves are unaffected even by large doses of TM 10. Doses of TM 10 which produce effective blockade do not impair conduction along adrenergic nerve trunks; the drug must, therefore, act at, or close to, the nerve terminals. TM 10 prevents the output of noradrenaline from the spleen on stimulating the splenic nerves; but, in acute experiments, it does not influence the liberation of pressor amines from the stimulated suprarenals. Examination of some ethers related to TM 10 revealed no correlation between TM 10-like adrenergic blocking activity and local anaesthetic activity. The action of TM 10 on adrenergic nerves does not, therefore, seem to be accounted for by axonal block.

To investigate the effect of vagal blockade with atropine on nocturnal fall in peak expiratory flow rate 10 patients with asthma who had a diurnal variation in peak expiratory flow rate of greater than 20% were given 30 micrograms/kg of intravenous atropine or a placebo at 4 am and 4 pm. Vagal blockade caused significant bronchodilatation at 4 am and 4 pm (peak expiratory flow rate rose from 260 to 390 l/min at 4 am and 400 to 440 l/min at 4 pm) and significantly increased the pulse rate from 60 to 121 beats/minute at 4 am and from 76 to 122 beats/minute at 4 pm. Nocturnal asthma was almost totally reversed, implying that vagal mechanisms are fundamental in its pathophysiology. Other mechanisms--diurnal changes in plasma adrenaline concentration, the activity of non-adrenergic non-cholinergic nerves, and circadian rhythms of inflammatory mediator activity--may also be implicated.

Sympathetic vasoconstriction of cerebral vessels has been proposed to be a protective mechanism for the brain, limiting cerebral perfusion and microcirculatory pressure during transient increases in arterial pressure. To furnish direct neural evidence for this proposition, we aimed to develop a method for recording cerebral sympathetic nerve activity (SNA) from the superior cervical ganglion (SCG). We hypothesized that SNA recorded from the SCG increases during imposed hypertension, but not during hypotension. Lambs (n = 11) were anesthetized (alpha-chloralose, 20 mg.kg(-1).h(-1)) and ventilated. SNA was measured using 25-microm tungsten microelectrodes inserted into the SCG. Arterial blood pressure (AP) was pharmacologically raised (adrenaline, phenylephrine, or ANG II, 1-50 microg/kg iv), mechanically raised (intravascular balloon in the thoracic aorta), or lowered (sodium nitroprusside, 1-50 microg/kg iv). In response to adrenaline (n = 10), mean AP increased 135 +/- 10% from baseline (mean +/- SE), and the RMS value of SNA (Square Root of the Mean of the Squares, SNA(RMS)) increased 255 +/- 120%. In response to mechanically induced hypertension, mean AP increased 43 +/- 3%, and SNA(RMS) increased 53 +/- 13%. Generally, (9 of 10 animals), SNA(RMS) did not increase, as AP was lowered with sodium nitroprusside. Using a new model for direct recording of cerebral SNA from the SCG, we have demonstrated that SNA increases in response to large induced rises, but not falls, in AP. These findings furnish direct support for the proposed protective role for sympathetic nerves in the cerebral circulation.

Catechol-O-methyl transferase (COMT) metabolizes a variety of catecholamines such as dopamine, adrenaline and noradrenaline. It exists in common high and low activity forms. The low activity form is the result of an amino acid substitution (val-108-met) which reduces the thermostability of the enzyme [Lotta et al. (1994) Biochemistry, 34, 4202-4210]. We have genotyped this polymorphism in 178 trios consisting of Han Chinese schizophrenic subjects and their parents in order to test the hypothesis that the high activity allele is transmitted more often to affected subjects. The data were analysed using the transmission disequilibrium test (TDT), a robust method of detecting linkage in the presence of allelic associations. Of the 131 parents heterozygous at this locus, 80 transmitted the high activity allele (val-108) to affected offspring, while the remaining 51 transmitted the low activity allele (p = 0.005, one-tailed). Combining this result with that of a previous TDT study of the same polymorphism in familial schizophrenia [Kunugi et al. (1996) submitted] gives significant evidence for linkage disequilibrium (p = 0.0015). However, val-108 is frequent in the Han Chinese population, and in the present sample, 239 of the 350 non-transmitted parental alleles were val-108 (68%). It is therefore unlikely that val-108 allele of COMT has a major effect on susceptibility to schizophrenia. Our results suggest that either val-108 is a minor risk factor for schizophrenia, that the COMT gene has additional polymorphisms with greater effect on risk, or that this region of chromosome 22 contains a susceptibility gene which is in linkage disequilibrium with the COMT gene.

1 Intracellular recordings were made from neurones in the myenteric plexus of the ileum removed from guinea-pigs. The effects of clonidine and adrenaline on membrane potential and resistance were observed.2 Clonidine (100 pM-30 nM) caused a concentration-dependent membrane hyperpolarization associated with a fall in neurone input resistance.3 The amplitude of the clonidine hyperpolarization, but not the conductance increase, was greater in cells with lower resting potentials and smaller in more polarized neurones. In a given cell, membrane hyperpolarization decreased and membrane depolarization increased the clonidine effect.4 Low potassium solutions enhanced and high potassium solutions reduced the hyperpolarizing action of clonidine but did not significantly change the conductance increase caused by clonidine.5 The concentration-effect curve for clonidine was displaced to the left when the extracellular calcium concentration was reduced. Conversely, clonidine was almost ineffective in elevated calcium concentrations. This was true for both the hyperpolarization and the conductance increase.6 It is suggested that clonidine activates a potassium conductance by causing an elevation in the free intracellular calcium concentration.7 Clonidine reversibly depressed the amplitude of the nicotinic fast excitatory postsynaptic potential and the noncholinergic slow excitatory postsynaptic potential.8 All the effects of clonidine were shared by adrenaline and the actions of both were reversed or prevented by phentolamine (100 nM-1 muM).

An investigation has been made into the mode and specificity of action of ventral medullary pressor neurones. These were activated by microinjections of excitant amino acid into the ventral brain surface of chloralose-anaesthetized, artificially ventilated cats, and a number of autonomic responses were measured. Indirect assessment of cardiac output (by CO2 delivery to the lungs) suggested that it was either unchanged or fell during pressor responses. The inference that activating the pressor neurones caused vasoconstriction was confirmed directly for hindlimb and mesenteric vascular beds, by a rise in inflow pressure when they were perfused at constant flow. Sympathetic activity also increased in cervical, splanchnic and inferior cardiac nerves. Bradycardia often (but not always) accompanied pressor responses, but this was abolished by vagotomy, although not by cutting the sinus and aortic nerves. In vagotomized cats, tachycardia could be produced during pressor responses even after either bilateral adrenalectomy or removal of the stellate ganglia, indicating both direct sympathetic drive to the heart and release of adrenal catecholamines. Plasma adrenaline levels were measured and found to increase by up to 20.2 times control values, plasma noradrenaline up to 12.6 times, and dopamine by a smaller amount. Activating ventral medullary pressor neurones appeared to have no significant action on pupils, nictitating membranes or piloerection. In three adrenalectomized, vagotomized cats, only small, inconsistent effects were measured on intestinal motility following pressor neurone excitation. However, large electrodermal responses could be evoked from the ventral medulla, but from a distinct area medial to the pressor neurones.(ABSTRACT TRUNCATED AT 250 WORDS)

Employing double-isotope derivative techniques, noradrenaline and adrenaline have been determined in plasma and in urine and dopamine in urine in 21 patients with essential hypertension as well as in 32 controls. Plasma noradrenaline rose with age in both groups of subjects. No differences were observed in plasma noradrenaline and plasma adrenaline in the resting supine position and in urinary excretion of noradrenaline and dopamine in hypertensive patients as compared to control subjects. Urinary excretion of adrenaline was somewhat lower in the hypertensives than in the controls. Treatment with alprenolol, a beta-adrenergic blocking agent, did not influence noradrenaline and adrenaline in plasma in the basal state or the urinary excretion of the three catecholamines. The combined treatment with alprenolol and hydralazine was followed by a significant rise in plasma noradrenaline. It is concluded that the adrenergic activity evaluated by circulating catecholamines is normal in most patients with essential hypertension.

A method for washing platelets by albumin density gradient separation, originally designed for the study of platelet coagulant activities, has been modified for platelet aggregation and metabolic studies. Platelets are sedimented into a continuous density gradient of isosmolar albumin containing apyrase to protect them from clumping and physical injury and are resuspended in calcium-free Tyrode's solution. The mean recovery of platelets after two separations relative to platelet-rich plasma (PRP) was 90.3%. When small amounts of plasma were added to washed platelet suspensions, aggregation and release of [14C]5-hydroxytryptamine (5HT) in response to adenosine diphosphate (adp) or 5HT were similar to results obtained with PRP. When fibrinogen was substituted for plasma, ADP-induced aggregation occurred but was feeble. Without added plasma or fibrinogen, platelets were refractory to ADP and insensitive to the cyclic endoperoxide analogue U44619. When both ADP and U44619 were added simultaneously, in low concentrations, to washed platelets without added plasma or fibrinogen, aggregation occurred immediately. Washed platelets were not aggregated by adrenaline, which potentiated ADP-induced aggregation. Several biochemical measurements which are sensitive indicators of cellular damage were normal in washed platelets, including [14C]adenine uptake, adenylate energy charge, hypoxanthine formation and the response of adenylate cyclase to stimulation by PGE1 or PGD2. Platelet coagulant activities were not made available and heparin-neutralizing activity (HNA) was not spontaneously released by the washing procedure, but the washed platelets responded normally to appropriate agents by developing coagulant activities and releasing HNA. The ultrastructure of washed platelets was similar to those in control PRP. Inclusion of apyrase in the first albumin gradient had a beneficial effect on platelet morphology, aggregation and metabolism, but washing at 37degreesC compared with 25degreesC did not. Albumin density gradient separation is a useful method for isolating platelets for aggregation and metabolic studies.

1. In the perfused cat submandibular gland efflux and influx of (45)Ca, and concentrations of K, (40)Ca and Mg in the effluent from the gland were measured under different experimental conditions.2. When the standard perfusion fluid was shifted to a high Mg (5 mM) or a low Ca (0.25 mM) solution the efflux of (45)Ca from the pre-labelled gland declined. The magnitude and the duration of the effect of the high Mg concentration was more marked at a low external Ca concentration and was abolished by Mersalyl (1 mM). When the standard perfusion fluid was shifted to a Mg-free solution the efflux of (45)Ca from the pre-labelled gland increased.3. After shift of (45)Ca containing perfusion fluid from normal to a high Mg (5 mM) solution the influx of (45)Ca to the gland increased rapidly.4. Both acetylcholine (ACh) and adrenaline caused a marked increase in the efflux of (45)Ca from the pre-labelled gland. This increase in efflux was also seen under conditions where the gland was unable to secrete, i.e. during perfusion with Ca-free and Na-free tetraethylammonium Locke solutions.5. Stimulation with ACh failed to reveal any rapidly occurring increase in influx of (45)Ca.6. Stimulation with ACh evoked a small temporary increase in the concentration of (40)Ca. and Mg in the effluent.7. It is suggested that Ca uptake by intracellular Ca-accumulating systems of the submandibular gland depends on the external Mg concentration and that ACh and adrenaline cause a release of Ca bound intracellularly.

1. Intracellular micro-electrode recordings were made from surface acini of mouse exorbital lacrimal glands placed in a Perspex bath through which oxygenated physiological saline solutions were circulated. Two micro-electrodes were inserted into neighbouring communicating cells. Through one of the electrodes, current pulses could be injected. The cells impaled were stimulated by iontophoresis of acetylcholine (ACh), adrenaline or isoprenaline from an extracellular micropipette. 2. During exposure to standard Krebs solution the resting membrane potential was -42.5 mV +/- 1.2 and the resting input resistance 3.3 Momega +/- 0.3. When the tips of the two intracellular micro-electrodes were more than 100 micrometer apart no electrical coupling between two impaled cells could be detected. At intertip distances below about 80 micrometer coupling was frequently observed. In all such cases the coupling ratio was 1. The resting current-voltage relation was almost linear within the membrane potential range of -30 to -80 mV. 3. During exposure to standard Krebs solution the resting membrane potential was -42.5 mV +/- 1.2 and the resting input resistance 3.3 Momega +/- 0.3. When the tips of the two intracellular micro-electrodes were more than 100 micrometer apart no electrical coupling between two impaled cells could be detected. At intertip distances below about 80 micrometer coupling was frequently observed. In all such cases the coupling ratio was 1. The resting current-voltage relation was almost linear within the membrane potential range of -30 to -80mV. 3. During exposure to standard Krebs solution short iontophoretic pulses of ACh or adrenaline caused fully reversible hyperpolarizations accompanied by marked reduction of surface cell membrane resistance and membrane time constant. The effects of ACh were blocked by atropine (1.4 x 10(-6)M). Iontophoresis of isoprenaline never had any detectable effect on membrane potential or resistance. 4. Applying de- or hyperpolarizing direct currents through one of the two intracellular micro-electrodes the effect of ACh or adrenaline could be observed at different lvels of resting potential. Depolarizing the acinar cell membrane resulted in an enhanced stimulant-evoked hyperpolarization whereas hyperpolarizing the acinar cell membrane resulted in a reduction, and at potentials more negative than -60 mV in a reversal of the stimulant-evoked potential change. The ACh equilibrium potential (EACh) under control conditions was -56.6 mV +/- 1.1 and EAdrenaline was -61.4 mV +/- 1.0. 5. Replacing the control superfusion solution by a Clfree sulphate solution resulted in an immediate shift of EACh towards more negative values. At steady state in the Cl-free solution the resting input resistance was 6.8 Momega +/- 1.3 EACh was -95.9 mV +/- 3.4. 6. Reducing [K]o from the usual 4.7 to 1.0 mM resulted in an immediate marked increase in the amplitude of ACh-evoked hyperpolarization whereas increasing [K]o to 10 mM almost abolished the ACh-evoked potential, but not resistance change. 7...

Explants and enzyme-dispersed cells of adrenal medulla from 10-12 day old rats were studied in culture for up to 3 weeks. Adrenomedullary chromaffin cells, nerve cells and satellite cells were clearly discernible. The nerve cells were few in number and did not show catecholaminespecific fluorescence. Chromaffin cells stored catecholamines, as judged by the Falck and Hillarp method, in varying amounts decreasing with age of the cultures and the distance from the explants. Exocytosis profiles observed with the electron microscope suggested that cultured chromaffin cells also released catecholamines. Moreover, the cells formed processes and frequently migrated into the outgrowth. After 6 days in culture, the great majority of chromaffin cells stored noradrenaline as revealed by electron microscopy with few adrenaline-storing cells being visible. Granular vesicles (approximately 80-240 nm in diameter) with cores of different electron densities were occasionally present in the same cell suggesting the occurrence of mixtures of primary and secondary amines. Apart from "chromaffin" granules, small clear and dense-cored vesicles (approximately 40-60 nm) were found both in the somata and cell processes. Chromaffin cells and their processes were often closely apposed and occasionally formed specialized attachment zones. As a whole, chromaffin cells in culture resembled small granule-containing cells in sympathetic ganglia. 0.5 mM dbcAMP prevented dedifferentiation of chromaffin cells as judged by the lack of processes, the size and amount of "chromaffin" granules and the high number of adrenaline-storing cells present after 6 days in culture. NGF caused a striking increase in the number of axons growing out from explants.