Nonlinear effects of potassium channel blockers on endothelium-dependent hyperpolarization.

In a number of published studies on endothelium-dependent hyperpolarization and relaxation, the results of the effects of K+ blockers have been difficult to interpret. When the effects of two blockers have been studied, often either blocker by itself had little effect, whereas the two blockers combined tended to abolish the responses. Explanations suggested in the literature include an unusual pharmacology of the K+ channels, and possible blocker binding interactions. In contrast, when we applied the same blockers to segments of small blood vessels under voltage clamp, the blockers reduced the endothelium-dependent K+ current in a linearly additive manner. Resolution of these contrasting results is important as endothelium-derived hyperpolarization (EDH) makes its greatest contribution to vasorelaxation in arterioles and small resistance arteries, where it can exert a significant role in tissue perfusion and blood pressure regulation. Furthermore, EDH is impaired in various diseases. Here, we consider why the voltage-clamp results differ from earlier free-running membrane potential and contractility studies. We fitted voltage-clamp-derived current-voltage relationships with mathematical functions and considered theoretically the effects of partial and total block of endothelium-derived K+ -currents on the membrane potential of small blood vessels. When the K+ -conductance was partially reduced, equivalent to applying a single blocker, the effect on EDH was small. Total block of the endothelium-dependent K+ conductance abolished the hyperpolarization, in agreement with various published studies. We conclude that nonlinear summation of the hyperpolarizing response evoked by endothelial stimulation can explain the variable effectiveness of individual K+ channel blockers on endothelium-dependent hyperpolarization and resulting relaxation.

Paradoxical effect of gonadotrophin-inhibiting hormone to negatively regulate neuropeptide Y neurones in mouse arcuate nucleus.

Regulation of reproduction and energy homeostasis are linked, although our understanding of the central neural mechanisms subserving this connection is incomplete. Gonadotrophin-inhibiting hormone (GnIH) is a neuropeptide that negatively regulates reproduction and stimulates food intake. Neuropeptide Y (NPY) and products of the pro-opiomelanocortin (POMC) precursor (ß-endorphin melanocortins) are appetite regulating peptides produced in the neurones of the arcuate nucleus; these peptides also regulate reproduction. In the present study, we determined the effects of GnIH on NPY and POMC neurones. Using brain slices from mice with transgenes for fluorescent tags in the two types of neurone and patch clamp electrophysiology, a predominant inhibitory effect of GnIH was observed. GnIH (100 nM) inhibited the firing rate in POMC cells, confirming the results of previous studies and consistent with the stimulatory effect of GnIH on food intake. Paradoxically (i.e. because both GnIH and NPY stimulate food intake), GnIH also had a predominantly inhibitory effect on action potential activity in NPY cells. GnIH also inhibited the secretion of NPY and α-melanocyte-stimulating hormone secretion in incubated hypothalamic blocks. GnIH (100 ng) injected into the cerebral ventricles of mice did not increase the number of NPY cells that were positively immunostained for c-Fos. Finally, dual label immunocytochemistry showed that 20% of NPY neurones had close contacts from GnIH fibres/varicosities. In conclusion, we confirm a negative effect of GnIH on POMC cells and demonstrate a paradoxical reduction of electrophysiological and functional activity in NPY cells.

Glucocorticoid treatment does not alter early cardiac adaptations to growth restriction in preterm sheep fetuses.

OBJECTIVE: To study the consequences of glucocorticoid treatment in fetal growth restriction (FGR) on cardiac function. SETTING: Laboratory. SAMPLE: Sheep. METHODS: Growth restriction was induced in sheep fetuses using single umbilical artery ligation (SUAL) on days 105-110 of gestation (term 147). Control fetuses were not ligated. Betamethasone (BM) (11.4 mg intramuscularly) or saline was administered to ewes on days 5 and 6 after surgery. Ewes were anaesthetised on day 7, the fetuses were removed, and their hearts were mounted on a Langendorff apparatus. Balloon catheters were inserted into the right and left ventricles. OUTCOME MEASURES: Ventricular contractile function and infarct area following ischaemia/reperfusion. RESULTS: The SUAL resulted in FGR (body weight 77% of control). The FGR was associated with increases in basal left ventricular pressure development and rates of contraction and relaxation. Right ventricular contraction was unaffected. Following brief ischaemia/reperfusion, the infarct area in FGR hearts was increased four-fold compared with controls. Antenatal BM resulted in a proportional increase in heart size and coronary flow, especially in FGR fetuses, and left ventricular pressure and heart rate responses to ß-adrenoceptor activation were increased. CONCLUSIONS: Fetal hearts rapidly adapt to FGR to maintain substrate delivery to the brain and heart. The FGR greatly enhanced the area of ischaemia, with implications for susceptibility in postnatal life. Antenatal BM treatment does not interfere with these cardiac changes but appears to increase left ventricle ß-adrenoceptor responsiveness, which may render the offspring vulnerable to subsequent cardiac dysfunction.

Effects of birth asphyxia on neonatal hippocampal structure and function in the spiny mouse.

Studies of human neonates, and in animal experiments, suggest that birth asphyxia results in functional compromise of the hippocampus, even when structural damage is not observable or resolves in early postnatal life. The aim of this study was to determine if changes in hippocampal function occur in a model of birth asphyxia in the precocial spiny mouse where it is reported there is no major lesion or infarct. Further, to assess if, as in human infants, this functional deficit has a sex-dependent component. At 37 days gestation (term=39 days) spiny mice fetuses were either delivered immediately by caesarean section (control group) or exposed to 7.5min of in utero asphyxia causing systemic acidosis and hypoxia. At 5 days of age hippocampal function was assessed ex vivo in brain slices, or brains were collected for examination of structure or protein expression. This model of birth asphyxia did not cause infarct or cystic lesion in the postnatal day 5 (P5) hippocampus, and the number of proliferating or pyknotic cells in the hippocampus was unchanged, although neuronal density in the CA1 and CA3 was increased. Protein expression of synaptophysin, brain-derived neurotrophic factor (BDNF), and the inositol trisphosphate receptor 1 (IP(3)R1) were all significantly increased after birth asphyxia, while long-term potentiation (LTP), paired pulse facilitation (PPF), and post-tetanic potentiation (PTP) were all reduced at P5 by birth asphyxia. In control P5 pups, PPF and synaptic fatigue were greater in female compared to male pups, and after birth asphyxia PPF and synaptic fatigue were reduced to a greater extent in female vs. male pups. In contrast, the asphyxia-induced increase in synaptophysin expression and neuronal density were greater in male pups. Thus, birth asphyxia in this precocial species causes functional deficits without major structural damage, and there is a sex-dependent effect on the hippocampus. This may be a clinically relevant model for assessing treatments delivered either before or after birth to protect this vulnerable region of the developing brain.

Role of mitochondria in contraction and pacemaking in the mouse uterus.

BACKGROUND AND PURPOSE: Uterine spontaneous contraction and pacemaking are poorly understood. This study investigates the role of the mitochondrial Ca(2+) store in uterine activity. EXPERIMENTAL APPROACH: We investigated the effects of mitochondrial and sarco-endoplasmic reticulum (SER) inhibitors on contraction, membrane potential (Vm) and cytosolic Ca(2+) concentration ([Ca(2+) ](c) ) in longitudinal smooth muscle of the mouse uterus. KEY RESULTS: The mitochondrial agents rotenone, carbonylcyanide-3-chlorophenylhydrazone (CCCP), 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP37157) and kaempferol decreased the force of contractions. The ATP synthase inhibitor oligomycin had no significant effect. The effects of these agents were compared with those of SER inhibitors cyclopiazonic acid (CPA), 2-amino ethoxyphenylborate (2-APB) and caffeine. All agents, except CPA and oligomycin, decreased contractile force. CPA and CCCP transiently increased contraction frequency, which returned to control levels, whereas rotenone, CGP37157, kaempferol and 2-APB decreased frequency and caffeine had no significant effect. Application of the mitochondrial agents when CPA functionally inhibited stores did not change contraction frequency but, with the exception of kaempferol, decreased force. CCCP caused depolarization and maintained increase in [Ca(2+) ](c) or depolarization/transient hyperpolarization and transient increase in [Ca(2+) ](c) for oestrus and di-oestrus tissues respectively. Rotenone caused hyperpolarization and maintained increase in [Ca(2+) ](c) . CGP37157 and kaempferol caused hyperpolarization but no measurable change in [Ca(2+) ](c) . Application of a range of K(+) channel blockers indicated a role of Ca(2+) -activated K(+) (K(Ca) ) channels in the CCCP- and CGP37157-induced actions. CONCLUSIONS AND IMPLICATIONS: Mitochondria have a modulatory role on uterine contractions, with mitochondrial inhibition reducing contraction amplitude and pacemaker frequency by changes in Vm, [Ca(2+) ](c) and/or Ca(2+) influx.

Early vitamin E supplementation attenuates diabetes-associated vascular dysfunction and the rise in protein kinase C-beta in mesenteric artery and ameliorates wall stiffness in femoral artery of Wistar rats.

AIMS/HYPOTHESIS: The impact of early vitamin E supplementation on vascular function in diabetes remains unresolved. Therefore, we examined the effects of vitamin E on functional and structural parameters and on chemical markers that are disturbed in diabetes in mesenteric and femoral arteries. METHODS: Segments of both arteries, taken from control and 8-week-old streptozotocin diabetic Wistar rats that were treated or not with vitamin E, were mounted on wire and pressure myographs, after which endothelium-dependent and -independent vasodilation was assessed. Passive mechanical wall properties and the localisation and levels of protein kinase C (PKC)-beta(2) and AGE were evaluated in these vessels. RESULTS: Vitamin E supplementation was associated with improved endothelium-dependent and -independent vasodilatation in mesenteric arteries from diabetic rats. Impaired endothelium-dependent vasodilatation in diabetic mesenteric vessels was associated with PKC-beta(2) up-regulation and this was prevented by vitamin E supplementation. Increased AGE accumulation and plasma isoprostane levels in diabetic rats were not changed by vitamin E. In the femoral artery, vitamin E supplementation had no effect on endothelium-dependent or -independent vasodilatation, but did prevent the wall stiffening associated with diabetes. CONCLUSIONS/INTERPRETATION: Early vitamin E supplementation has a beneficial effect on diabetes-induced endothelial dysfunction in resistance arteries. This benefit may arise from a direct effect on smooth muscle function, as a result of inhibition of the PKC-beta(2) isoform by vitamin E.

How important is stimulation of alpha-adrenoceptors for melatonin production in rat pineal glands?

The objective of this study was to determine the role of alpha-adrenoceptors in melatonin production by rat pineal gland. Pineal glands were isolated from adult male rats and maintained in organ baths. The perfusate was sampled every 5 min, stored, and later assayed for melatonin. Exposure to norepinephrine (10 microM) or the beta-adrenoceptor agonist orciprenaline (2-10 microM) increased the glands' production of melatonin. The time courses of melatonin production in response to these agonists were unaffected by the rats' pretreatment in vivo with the alpha-adrenoceptor antagonist prazosin (2 mg/kg i.p., three times). Rats that had had their superior cervical ganglia removed were primed with either orciprenaline (2 mg/kg i.p) or both orciprenaline and phenylephrine (1 mg/kg i.p) 1 hr before decapitation. Exposure of the pineal glands from these rats to orciprenaline evoked melatonin release that was similar in each group. These results lend weight to the suggestion that the marked potentiation by alpha-adrenoceptor agonists of the stimulation of cAMP and N-acetyltransferase (NAT) by beta-adrenoceptor agonists, demonstrated most readily in cultured glands or dispersed rat pinealocytes, does not carry over into significant augmentation of melatonin production in intact pineal glands.

Glycyrrhetinic derivatives inhibit hyperpolarization in endothelial cells of guinea pig and rat arteries.

Glycyrrhetinic acid (GA) derivatives have been used to implicate gap junctions in vasorelaxation attributed to endothelium-derived hyperpolarizing factor (EDHF). The aim of this study was to assess whether GA compounds affect endothelial cell hyperpolarization. Membrane potentials were recorded from dye-identified endothelial and smooth muscle cells of guinea pig coronary and rat mesenteric arteries. GA derivatives had varied effects on the resting membrane potential: depolarization, hyperpolarization, or no effect, depending on the artery. 18alpha-GA (50 microM) had a small variable effect on ACh-induced hyperpolarizations in endothelial cells. 18beta-GA (30 microM) and carbenoxolone (100 microM) significantly reduced ACh-induced hyperpolarizations in both endothelial and smooth muscle cells. Smooth muscle action potentials in rat tail arteries were smaller and slower in the presence of 18beta-GA. Nerve-induced excitatory junction potentials were inhibited by 18beta-GA and carbenoxolone, whereas the time course of their decay initially increased and then decreased. In conclusion, the GA compounds had a range of effects. Their inhibition of the EDHF hyperpolarization and relaxation in the smooth muscle may stem from the inhibition of endothelial cell hyperpolarization.

EDHF is not K+ but may be due to spread of current from the endothelium in guinea pig arterioles.

Endothelium-derived hyperpolarizing factor (EDHF)-attributed hyperpolarizations and relaxations were recorded simultaneously from submucosal arterioles of guinea pigs with the use of intracellular microelectrodes and a video-based system, respectively. Membrane currents were recorded from electrically short segments of arterioles under single-electrode voltage clamp. Substance P evoked an outward current with a current-voltage relationship that was well described by the Goldman-Hodgkin-Katz equation for a K+ current, consistent with the involvement of intermediate- and small-conductance Ca2+-activated K+ channels. 1-Ethyl-2-benzimidazolinone relaxed the arterioles and evoked hyperpolarizations that were blocked by charybdotoxin, but not by iberiotoxin. Application of K+ induced depolarization under conditions in which EDHF evoked hyperpolarization. The Ba2+-sensitive component of the K+-induced current was inwardly rectifying, in contrast to the outwardly rectifying current evoked by substance P. EDHF-attributed hyperpolarizations in dye-identified smooth muscle cells were indistinguishable from those recorded from dye-identified endothelial cells in the same arterioles. These results provide evidence that EDHF is not K+ but may involve electrotonic spread of hyperpolarization from the endothelial cells to the smooth muscle cells.

K+ currents underlying the action of endothelium-derived hyperpolarizing factor in guinea-pig, rat and human blood vessels.

Membrane currents attributed to endothelium-derived hyperpolarizing factor (EDHF) were recorded in short segments of submucosal arterioles of guinea-pigs using single microelectrode voltage clamp. The functional responses of arterioles and human subcutaneous, rat hepatic and guinea-pig coronary arteries were also assessed as changes in membrane potential recorded simultaneously with contractile activity. The current-voltage (I-V) relationship for the conductance due to EDHF displayed outward rectification with little voltage dependence. Components of the current were blocked by charybdotoxin (30-60 nM) and apamin (0.25-0.50 microM), which also blocked hyperpolarization and prevented EDHF-induced relaxation. The EDHF-induced current was insensitive to Ba2+ (20-100 microM) and/or ouabain (1 microM to 1 mM). In human subcutaneous arteries and guinea-pig coronary arteries and submucosal arterioles, the EDHF-induced responses were insensitive to Ba2+ and/or ouabain. Increasing [K+]o to 11-21 mM evoked depolarization under conditions in which EDHF evoked hyperpolarization. Responses to ACh, sympathetic nerve stimulation and action potentials were indistinguishable between dye-labelled smooth muscle and endothelial cells in arterioles. Action potentials in identified endothelial cells were always associated with constriction of the arterioles. 18beta-Glycyrrhetinic acid (30 microM) and carbenoxolone (100 microM) depolarized endothelial cells by 31 +/- 6 mV (n = 7 animals) and 33 +/- 4 mV (n = 5), respectively, inhibited action potentials in smooth muscle and endothelial cells and reduced the ACh-induced hyperpolarization of endothelial cells by 56 and 58 %, respectively. Thus, activation of outwardly rectifying K+ channels underlies the hyperpolarization and relaxation due to EDHF. These channels have properties similar to those of intermediate conductance (IKCa) and small conductance (SKCa) Ca2+-activated K+ channels. Strong electrical coupling between endothelial and smooth muscle cells implies that these two layers function as a single electrical syncytium. The non-specific effects of glycyrrhetinic acid precludes its use as an indicator of the involvement of gap junctions in EDHF-attributed responses. These conclusions are likely to apply to a variety of blood vessels including those of humans.

EDHF, NO and a prostanoid: hyperpolarization-dependent and -independent relaxation in guinea-pig arteries.

The contribution of endothelium-derived hyperpolarizing factor (EDHF), nitric oxide (NO) and a prostanoid (PG) to endothelium-dependent hyperpolarization and relaxation were assessed in coronary and mammary arteries of guinea-pigs by integration of the responses evoked during discrete applications of acetylcholine (ACh). The results of this integration approach were compared with those using traditional peak analysis methods. N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM) and indomethacin (1 microM), alone or in combination, were without effect on peak hyperpolarizations or relaxations while they markedly reduced the integrated responses in both arteries. Integrated responses attributed to NO and PG were larger than those attributed to EDHF in the coronary artery (at 2 microM ACh, hyperpolarization (mV s): NO, 4200+/-91; PG, 5046+/-157; EDHF, 1532+/-94; relaxation (mN s mm(-1)): NO, 2488+/-122; PG, 2234+/-96; EDHF, 802+/-54). Integrated responses attributed to NO, PG and EDHF were similar in the mammary artery (at 2 microM ACh, hyperpolarization: NO, 347+/-69; PG, 217+/-49; EDHF, 310+/-63; relaxation: NO, 462+/-94; PG, 456+/-144; EDHF, 458+/-40). Gilbenclamide (1 microM) all but abolished the hyperpolarization attributable to NO and PG but not EDHF in both arteries allowing assessment of the role of the hyperpolarization in relaxation. Gilbenclamide was without effect on the integrated relaxation due to NO but significantly reduced the relaxation associated with PG in the two arteries. In conclusion, integration of the responses enabled a more complete assessment of the contribution of EDHF, NO and PG to endothelium-dependent responses, which were strikingly different in the two arteries. There is commonality in the role of hyperpolarization in relaxation in both arteries: EDHF-dependent relaxation is strongly dependent on hyperpolarization; hyperpolarization plays an important role in PG relaxation, whereas it has a small facilitatory role in NO-dependent relaxation.

Changes in the mechanisms involved in uterine contractions during pregnancy in guinea-pigs.

1. The mechanisms involved in contraction in guinea-pig myometrium were compared at mid- and late pregnancy. Tension was recorded simultaneously with either membrane potential or cytoplasmic calcium ([Ca2+]i) in strips exposed briefly to prostaglandin F2alpha (PGF). 2. PGF-induced increases in tension were underpinned by action potentials followed by sustained depolarization and biphasic increases in [Ca2+]i at mid- (peak, 879 +/- 199 nM; sustained, 298 +/- 35 nM, n = 11) and late pregnancy (peak, 989 +/- 302 nM; sustained 178 +/- 33 nM, n = 8). 3. At mid- and late pregnancy, nifedipine (10-6 M) reduced (a) the PGF-induced increase in tension to 84 and 35 %, (b) the level attained during the depolarization by 2 and 12 mV and (c) the peak rise in [Ca2+]i to 42 and 17 %. The sustained rises in [Ca2+]i were resistant to nifedipine. 4. In Ca2+-free solution (containing 1 mM EGTA), PGF elicited an increase in tension that was 26 % of that in 2.5 mM Ca2+ and an increase in [Ca2+]i (24 % of the sustained level) at mid-pregnancy but no increase in tension or [Ca2+]i at term. 5. At both stages of pregnancy, PGF decreased the level of [Ca2+]i required to elicit increases in tension comparable to those evoked by high K+o. The slope of the tension-[Ca2+]i curves were steeper in mid- than in late pregnancy. 6. In conclusion, at mid-pregnancy, the contractile response of the guinea-pig myometrium to PGF involves Ca2+ influx through L-type voltage-operated Ca2+ channels (VOCCs) and by receptor-operated mechanisms, release of Ca2+ from intracellular stores, and an increase in the sensitivity of the contractile apparatus to Ca2+. At term the situation is different: a modest increase in the sensitivity of the contractile apparatus to Ca2+ persists and there is a major reliance on Ca2+ influx through VOCCs.

Contractile activity, membrane potential, and cytoplasmic calcium in human uterine smooth muscle in the third trimester of pregnancy and during labor.

OBJECTIVE: This study was undertaken to investigate in human tissue samples the mechanisms underlying spontaneous and prostaglandin F(2)(alpha)-induced contractions during the final trimester of pregnancy and labor. STUDY DESIGN: Membrane potential and cytoplasmic calcium were recorded simultaneously with contraction in uterine strips obtained from the lower segment during cesarean delivery. RESULTS: Between week 28 of gestation and term there was a progressive increase in the frequency of spontaneous contractions and a decrease in the negative potential of the membrane. The response to prostaglandin F(2alpha) was biphasic. The initial excitatory component remained stable toward term. A later inhibitory component, which was underpinned by increased activity of the sodium-potassium adenosine triphosphatase pump, decreased at the time of labor. CONCLUSIONS: There is a gradual increase in excitability in uterine muscle throughout the third trimester of human pregnancy. The initial component of the prostaglandin response is a large contraction that is kept brief by a subsequent inhibitory component of the response, which ensures that full relaxation occurs between contractions.

Endothelium-dependent hyperpolarization in resting and depolarized mammary and coronary arteries of guinea-pigs.

1. The membrane potential responses in guinea-pig coronary and mammary arteries attributable to endothelium-derived nitric oxide (NO), prostaglandin (PG) and hyperpolarizing factor (EDHF), and to exogenous NO and the prostacyclin analogue, iloprost, were compared at rest and when depolarized with the thromboxane analogue, U46619. 2. In the coronary artery, stimulation of the endothelium with acetylcholine (ACh) evoked hyperpolarization attributable to NO and a PG with similar pD2s at rest and in the presence of U46619. However, in depolarized tissues, the pD2 of the response attributed to EDHF required a 10 fold lower concentration of ACh compared with at rest. 3. In the mammary artery, lower concentrations of ACh were required to evoke NO- and EDHF-dependent hyperpolarizations in depolarized mammary artery compared with at rest, while PG-dependent hyperpolarization did not occur until the concentration of ACh was increased some 10 fold both at rest and in U46619. 4. The smooth muscle of the coronary artery of guinea-pigs was some 4 fold more sensitive to exogenous NO and iloprost than was the mammary artery. 5. In conclusion, the membrane potential response in arteries at rest, that is, in the absence of constrictor, may be extrapolated to events in the presence of constrictor when NO and PG are under study. However, the sensitivity to ACh and the magnitude of the hyperpolarization attributed to EDHF obtained in tissues at rest may underestimate these parameters in depolarized tissues.

Hyperpolarization and slowing of the rate of contraction in human uterus in pregnancy by prostaglandins E2 and f2alpha: involvement of the Na+ pump.

1. The effects of prostaglandins E2 (PGE) and F2alpha (PGF) on membrane potential and isometric tension and cytoplasmic free calcium concentration ([Ca2+]i) and tension were studied in strips of uterine smooth muscle obtained from women undergoing Caesarean delivery at term and during established labour. 2. Prostaglandins (PGs) evoked a biphasic response. The excitatory component consisted of depolarization of the membrane, which initiated spike action potentials, an increase in [Ca2+]i and tension development. The membrane remained depolarized at -19 +/- 1 mV for about 2 min, then repolarized abruptly, [Ca2+]i promptly returned to basal levels, and tension development ceased. 3. This component of the response to PGE or PGF was followed by a slow hyperpolarization which reached -85 +/- 2 mV (n = 22) at term and -70 +/- 2 mV (n = 9) during labour, and during which spontaneous action potentials and tension development did not occur. 4. Nifedipine (10-6 M) abolished spontaneous activity, abolished PG-induced action potentials and reduced the increase in [Ca2+]i (9 +/- 3 %, n = 6), the depolarization (10 +/- 1 mV, n = 14), the tension (2 +/- 1 %, n = 14) and the hyperpolarization (9 +/- 1 mV, n = 14, at term). 5. A variety of K+ channel blockers were without effect on the peak amplitude of the PG-induced hyperpolarization but the latter did not occur in the presence of ouabain (10-6 M) or in K+-free or low-Na+ solutions, suggesting an involvement of the Na+-K+-ATPase pump. 6. In conclusion, a substantial dependence on Ca2+ influx through voltage-operated Ca2+ channels accounts for the importance of membrane potential in regulating contractions in human uterine smooth muscle. The classical excitatory effect of PGE and PGF is followed by hyperpolarization involving the Na+-K+-ATPase pump. The hyperpolarization restricts the response to a single contraction and decreases the frequency of subsequent contractions. The amplitude of the hyperpolarization decreases during labour, allowing contraction frequency to increase. Its persistence at this time ensures complete relaxation between each single robust contraction, preventing spasm of the uterus that would restrict blood flow to the fetus during delivery.

Muscarinic receptor activation in guinea-pig chromaffin cells causes decreased membrane conductance and depolarization.

Membrane potentials were recorded with conventional intracellular microelectrodes from chromaffin cells in isolated, bisected adrenal glands from guinea-pigs. The local pressure ejection of muscarinic agonists, acetylcholine (in the presence of hexamethonium) or bethanecol, caused a transient depolarization that was relatively slow (1-2 s) in onset compared with the depolarization associated with the activation of nicotinic receptors. Muscarinic receptor-induced depolarization was associated with an increase in input resistance and the firing of action potentials. Repetitive stimulation of splanchnic nerve fibers within the gland, in the presence of hexamethonium, caused a maintained depolarization that was slow in both onset and decay and in many cells caused the repetitive firing of action potentials. It is suggested that, in this species, the exocytosis of catecholamines caused by the activation of muscarinic receptors, described by others, may be due to the initiation of tetrodotoxin-sensitive action potentials and consequent opening of voltage sensitive Ca2+ channels.

Pregnancy-induced decrease in evoked excitatory junction potentials in guinea pig uterine artery.

The effects of stimulating the intramural nerves on the membrane potential and tension in the uterine artery of virgin guinea pigs were compared with the responses during pregnancy. In all tissues the amplitude of the excitatory junction potential (EJP) increased as the stimulus voltage was increased. The rate of increase in EJP amplitude in tissues from virgin animals greatly exceeded that recorded in late pregnant tissues. EJPs were abolished by tetrodotoxin but were resistant to blockade by alpha-adrenoceptor antagonists. Stimulation of the nerves also evoked a slow depolarization and contraction which were abolished by both tetrodotoxin and alpha-adrenoceptor antagonists. The amplitudes of the depolarizations and contractions were not correlated. The role of EJPs and alpha-adrenoceptor activation in the control of vascular function is discussed. Fluorescence histochemistry revealed a decrease in the density of the catecholamine innervation that was correlated with a decrease in catecholamine content as pregnancy progressed. In addition, there appeared to be a difference in the arrangement of the fluorescent varicosities, with a shift from varicosities that were close to the outer layer of smooth muscle in virgin tissues to those that were more distantly dispersed in the adventitia during late pregnancy. The changes would be expected to reduce the effectiveness of vasoconstrictor drive to the uterine artery as pregnancy progresses.

The association between melatonin production and electrophysiology of the guinea pig pineal gland.

Melatonin production by isolated pineal glands from guinea pigs was examined under conditions that affect membrane potential or the firing of action potential-like spikes. In glands from superior cervical ganglionectomized animals, depolarization resulting from increasing extracellular potassium concentration to 100 mM did not initiate melatonin production, and it delayed the response to the beta-adrenoceptor agonist orciprenaline. In glands from intact animals melatonin production was initiated by exposure to 100 mM potassium with a time-course similar to the response to orciprenaline. A proportion of this response was propanol resistant, suggesting that the normal control of melatonin production may involve a neurotransmitter in addition to norepinephrine. Exposure to verapamil or nifedipine, or removal of extracellular calcium, previously shown to eliminate action potential-like spikes, did not substantially affect the increase in melatonin production induced by orciprenaline. Phenylephrine, which stimulates spiking, produced only a slight increase in melatonin production. It is concluded that the depolarization and the spiking are not closely related to the stimulation of melatonin production, but may relate principally to the secretion of a substance other than melatonin.

Release of endothelium-derived hyperpolarizing factor (EDHF) by M3 receptor stimulation in guinea-pig coronary artery.

1. The muscarinic receptor subtype(s) involved in the release of endothelium-derived hyperpolarizing factor (EDHF) were studied in the guinea-pig coronary artery by recording the membrane potential in the smooth muscle cells with intracellular microelectrodes. 2. Acetylcholine (ACh, pD2 6.68) was 10 times more potent than the M2 agonist, oxotremorine (pD2 5.65) and 500 fold more potent than the M1 agonist, McN-A-343 (pD2 3.95) in evoking the EDHF hyperpolarization. 3. The M3 muscarinic antagonist, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) was the most potent (pA2 9.5) in inhibiting the release of EDHF evoked by ACh, being more potent than pirenzepine (pA2 6.7), and AFDX-116 (pA2 6.1) which preferentially block M1 and M2 receptors, respectively. 4. These results suggest that EDHF is released from the endothelium of the guinea-pig coronary artery upon the activation of the muscarinic M3 receptor subtype.