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.

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.

Multiple sites for the initiation of action potentials in neurons of the inferior mesenteric ganglion of the guinea-pig.

Responses to nerve stimulation were recorded with intracellular microelectrodes from neurons in the inferior mesenteric ganglion of the guinea-pig. An event was recorded which was subthreshold for a somatic action potential but which had a short rise time and a rapid initial repolarization that gave the event a small but well-defined peak. This event was termed a "partial spike." During repeated stimulation of the same nerve trunk, the fluctuations in the amplitude of the partial spike were small compared to those of evoked synaptic potentials. Stimulation of different nerve trunks evoked partial spikes of different amplitudes. When different nerve trunks were stimulated at short intervals between stimuli, one partial spike could occlude another partial spike. Antidromic responses could not be blocked by a preceding partial spike. This suggests that partial spikes are not initial segment spikes. Tubocurarine reversibly abolished partial spikes which is indicative of a synaptic origin. It is concluded that partial spikes result from action potentials initiated by synaptic potentials in the dendrites and which fail to generate somatic action potentials.

Response of the rat myometrium to phenylephrine in early pregnancy and the effects of 6-hydroxydopamine.

1. The contractile responses of the longitudinal and circular muscle layers of the rat uterus to the alpha 1-adrenoceptor agonist phenylephrine were measured on days 3-6 of gestation. There was a progressive increase in sensitivity to phenylephrine in both muscle layers between days 3 and 6 of gestation. Overall, this amounted to a 13 and 9 fold increase in sensitivity in longitudinal and circular muscles, respectively. In longitudinal muscle the slope of the Hill plot was 2 on day 3 of pregnancy and was decreased to 1 thereafter. 2. The sympathetic nerve terminals innervating the smooth muscle of the uterus were destroyed by administration of 6-hydroxydopamine (2 x 50 mg kg-1) 4-7 days before testing with phenylephrine. Following this treatment there was a significant increase in sensitivity to phenylephrine on day 3 in both muscle layers. After day 4, the longitudinal muscle was less sensitive to phenylephrine. 3. In the longitudinal muscle there was a progressive increase in the contractile response to maximal concentrations of phenylephrine and to high potassium (100 mM) between days 3 and 6 of pregnancy. In the circular muscle the responsiveness to both phenylephrine and potassium remained unchanged between days 3 and 6 of gestation. 6-Hydroxydopamine had no effect on the maximal responses to phenylephrine or high potassium in either muscle layer. 4. In conclusion, denervation supersensitivity of uterine smooth muscle following injection of 6-hydroxydopamine is observed only on day 3 of pregnancy and appears to be replaced by subsensitivity by day 6. The decrease in the slope of the Hill plot in longitudinal muscle after day 3 may be explained by changes in events between activation of alpha 1-adrenoceptors and contraction.

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.

Pilocarpine-induced relaxation of rat tail artery by a non-cholinergic mechanism and in the absence of an intact endothelium.

1. The partial muscarinic agonist, pilocarpine, evoked concentration-dependent relaxation with an EC50 of 2.4 x 10(-3) M in isolated segments of rat tail artery that were constricted with phenylephrine (10(-8) to 2 x 10(-7) M). Acetylcholine also evoked concentration-dependent relaxation but was more potent than pilocarpine (EC50, 6.5 x 10(-7) M). 2. The concentration-relaxation curves for pilocarpine were not affected by the muscarinic antagonists, atropine (10(-9) M) or pirenzepine (5 x 10(-7) M), while the concentration-relaxation curves for acetylcholine-evoked relaxation of the same tissues were shifted some 10 fold to the right by these concentrations of atropine and pirenzepine. 3. Acetylcholine failed to evoke relaxation following removal of the endothelium. The smooth muscle of the rat tail artery was some 10 fold more sensitive to the relaxing action of pilocarpine following denudation of the endothelium. 4. The effects of pilocarpine and acetylcholine on membrane potential were studied in tissues that were depolarized to -39 +/- 1 mV with phenylephrine (5 x 10(-8) to 2 x 10(-7) M). In intact tissues, pilocarpine caused hyperpolarization, an effect that persisted in the presence of muscarinic antagonists. Acetylcholine also evoked hyperpolarization. 5. Following removal of the endothelium, pilocarpine (10(-5) to 10(-3) M) evoked hyperpolarization in 6 of 15 preparations and a decrease in the frequency of action potentials in the remainder. Both of these responses were associated with relaxation. 6. The effects of pilocarpine were compared with other agents that evoke endothelium-independent relaxation. The concentration-relaxation curves in response to pilocarpine and nitroprusside were shifted to the right by ferricyanide (10-5 M) and methylene blue (10-5 M). Glibenclamide (10-6 M) was without effect on the hyperpolarization and relaxation evoked by pilocarpine (10' to 10- M).7. Thus, pilocarpine evokes relaxation of rat tail artery independently of the cholinergic system and it is suggested that this is achieved by decreasing the frequency of action potentials in the smooth muscle.

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.

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.

Currents caused by the spontaneous release of quanta of acetylcholine onto chromaffin cells in guinea-pig adrenal medulla.

Membrane potentials were recorded from chromaffin cells in isolated bisected adrenal glands from guinea-pigs. Spontaneous excitatory synaptic potentials (SESPs) were recorded whose frequency was increased following brief (up to 10 s) periods of presynaptic nerve stimulation at 10-30 Hz. The single electrode voltage-clamp method was used to record the currents underlying all but the largest SESPs. Spontaneous excitatory synaptic currents (SESCs) decayed according to a single exponential with a time constant of about 8 ms at 30 degrees C. Thus the neuronal nicotinic receptor-channels giving rise to SESCs in chromaffin cells are probably very similar to those that are opened by quanta of acetylcholine in sympathetic ganglion cells.

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.

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.

Induction of prolonged excitability in myometrium of pregnant guinea-pigs by prostaglandin F2 alpha.

1. Electrical and mechanical responses to prostaglandin F2 alpha (PGF) were studied in circular myometrium, with or without endometrium, during the first 3 weeks of gestation of the guinea-pig. 2. Muscle strips from which endometrium had been removed became inexcitable within 30-40 min of isolation from the animal such that action potentials and contraction could not be initiated by depolarizing current steps. Raising the concentration of potassium in the perfusing solution resulted in a small contraction. 3. In these preparations PGF induced complex action potentials that consisted of spikes and a prolonged plateau of depolarization. Each action potential was associated with a large phasic contraction. 4. Contractions induced by PGF are unlikely to result predominantly from release of calcium from intracellular stores since the ability of the agonist to evoke a response was reduced by some 97% in the absence of external calcium or in the presence of calcium channel blockers. 5. When preceded by a brief exposure to PGF, the contractile response to high potassium was enhanced to equal that in response to PGF. Enhancement persisted for approximately 30 min after removal of PGF and was not dependent upon the presence of external calcium. 6. Muscle strips with intact endometrium contracted spontaneously for hours. Each contraction was associated with a complex action potential, both of which were abolished by indomethacin. 7. It is concluded that PGF transforms inexcitable calcium channels in the membrane of the smooth muscle cells of the circular myometrium into excitable ones. The study also suggests that endogenous prostaglandin of endometrial origin may prevent the 'run-down' of channels in vivo.

Ionic mechanisms underlying action potentials in myometrium.

1. The ionic mechanisms underlying the simple spike action potential in longitudinal myometrium of pregnant rats and the complex action potential which occurs in the same layer of pregnant guinea-pigs are discussed. 2. The current during the upstroke of the simple spike is carried by calcium and repolarization results from inactivation of the calcium current and activation of a potassium current. 3. A slow inward current underlies the plateau component of the complex action potential and calcium is involved in carrying or regulating this current. 4. Single channel recordings from the longitudinal myometrium of pregnant guinea-pigs reveal large conductance (130-170 pS) potassium channels which are activated by depolarization of the membrane. The activation of these channels during the upstroke of the spike would contribute to the rapid termination of the spike. 5. The duration of the plateau component of the complex action potential closely correlates with the duration of contraction and it is suggested that sufficient calcium may enter the cell during the action potential to activate the contractile apparatus directly.

Single channel Cl- and K+ currents from cells of uterus not treated with enzymes.

Single channel currents were recorded from uterine smooth muscle cells of pregnant guinea-pigs. The cells were within small bundles of smooth muscle of low input resistance. Giga-ohm seals were formed between the patch clamp electrodes and the membrane of smooth muscle cells that had not been treated with enzymes. In cell-attached mode, outward current steps were observed that had a conductance of 130-170 pS, a reversal potential close to the potassium reversal potential, and the probability of a channel being in the open state increased e-fold per 8-11 mV depolarization. In the cell-free, inside-out patch mode, currents were recorded that had a large conductance, 420 pS, and smaller conductance levels. When the chloride concentration was changed the reversal potential shifted in a manner consistent with the Nernst equation indicating that the channels were permeable to chloride ions.

Melatonin production by the guinea pig pineal gland in vitro.

The fluid bathing pineal glands isolated from guinea pigs was collected serially and its melatonin content was estimated. Production was found to be high (500 pg/5 min) soon after isolation of the gland: it declined exponentially to a maintained lower level (50 pg/5 min) with a half-time of 16-20 min. A rapid increase could be produced by exposure of the gland to the beta-adrenoceptor agonist orciprenaline or by stimulating it electrically. The time course and the extent of the responses to either form of stimulation were similar in glands that had been taken in the morning, at dusk or in mid-dark: the prestimulation peak was lower from glands taken at dusk. Thus the pineal gland of the guinea pig is capable of responding rapidly to stimulation of its beta-adrenoceptors at any time. These responses parallel the depolarization observed intracellularly in this species when the pineal gland is stimulated electrically or exposed to beta-adrenoceptor agonists.

Gestational changes in the utilization of intracellularly stored calcium in the myometrium of guinea-pigs.

1. The ability of oxytocin and prostaglandin F2 alpha (PGF) to induce contraction in guinea-pig myometrium in calcium-free solution was studied in an attempt to assess the extent to which intracellular calcium stores could be released by these two agonists. Both longitudinal and circular muscle layers were studied separately and the effects of gestational age were also examined. 2. In longitudinal strips, the responses to oxytocin and PGF in the absence of external calcium decreased progressively throughout gestation. Responses of circular strips to both agonists were unchanged throughout pregnancy, until day 64, when no response to PGF could be elicited. 3. Pre-treatment with high potassium (and normal calcium) increased the responses to the agonists in calcium-free medium while pre-treatment with beta-adrenoceptor agonists had no effect on responses to oxytocin or PGF. 4. Responses to both agonists decreased with time in calcium-free solution suggesting a loss of calcium from stores with a half-time of 3 min. The rate of the decline in the responses was the same in both muscle layers and did not change with gestational age. 5. In the presence of lanthanum contractions evoked by oxytocin, but not PGF, were augmented 2-3-fold. This potentiation of the response to oxytocin occurred in both muscle layers and throughout gestation. 6. Each agonist evoked only one response in calcium-free solution containing EGTA. The response to PGF in longitudinal strips following a challenge with oxytocin was reduced, compared with the response to PGF when applied first while the response to oxytocin in these strips was unchanged following exposure to PGF. In circular strips neither oxytocin- nor PGF-induced contractions were altered following prior exposure to the other agonist. 7. It is concluded that oxytocin and PGF operate via two distinct mechanisms to release intracellularly stored calcium in both longitudinal and circular components of the guinea-pig myometrium and a hypothesis to explain the results is presented.

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.