Stimulation of beta-adrenoceptors inhibits calcium-dependent potassium-channels in mouse macrophages

K+ efflux in mouse macrophages exhibited a rate constant (kK) of 0.67 +/- 0.04 (h)-1 (mean +/- SEM of 16 experiments). This was strongly stimulated by increasing concentrations of the Ca2+ ionophore A23187 up to a maximal value of 4.01 +/- 0.25 (h)-1 with an IC50 of 7.6 +/- 1.9 microM (mean +/- SEM of 6 experiments). Similar results were obtained with the Ca2+ ionophore ionomycin. Binding experiments with 3H-dihydroalprenolol revealed a high density of beta-adrenergic receptors (97.5 +/- 5.2 fmol/mg protein) with apparent dissociation constant of 2.03 +/- 0.06 nM. Isoproterenol at a concentration of 10(-6)-10(-5) M induced a two- to threefold stimulation of endogenous levels of cyclic AMP (cAMP). A23187-stimulated K+ efflux was partially inhibited by stimulation of adenylate cyclase with isoproterenol, forskolin or, PGE1; exogenous cAMP; and inhibition of phosphodiesterase with MIX (1-methyl-3-isobutylxanthine). Maximal inhibition of K+ efflux was obtained by simultaneous addition of isoproterenol and MIX. In dose-response curves, the isoproterenol-sensitive K+ efflux was half-maximally inhibited (IC50) with 2-5 X 10(-10) M of isoproterenol concentration. Propranolol was able to completely block the effect of isoproterenol, with an IC50 of about 1-2 X 10(-7) M. Isoproterenol and MIX were also able to partially inhibit ionomycin-stimulated K+ efflux. Isoproterenol and MIX did not inhibit A23187-stimulated K+ efflux in an incubation medium where NaCl was replaced by sucrose (or choline), suggesting the involvement of an Na+:Ca2+ exchange mechanism. Our results show that stimulation of beta-adrenoceptors in mouse macrophages counterbalances the opening of K+ channels induced by the calcium ionophore A23187. This likely reflects a decrease in cytosolic free calcium content via a cAMP-mediated stimulation of Na+:Ca2+ exchange.     

Ion-mediated resistance to osmotic changes of ram spermatozoa: the role of amiloride and ouabain

The aim of this study was to evaluate whether the Na+/K+ and Na+/H+ exchange can maintain the function of fresh ram spermatozoa. We analyzed the quality parameters of spermatozoa from fresh ram ejaculates incubated in iso- (about 300 mOsm), hypo- (about 100 mOsm) and hyperosmotic (about 900 mOsm) media in the presence of ouabain a specific inhibitor of the Na+/K+ ATP-ase or amiloride, a specific inhibitor of the Na+/H+ antiporter. Ouabain increased the percentage of morphologically altered acrosomes in isoosmotic media (from about 10% to 15% in control to about 30% with 10(-4) M ouabain) and decreased the percentage of total motility (from about 80% in control to about 50% to 55% with 10(-4) M ouabain). Ouabain decreased the mean linearity component of motility and decreased the frequency of head displacement. The addition of ouabain increased the percentage of altered acrosomes in the hypo- and hyperosmotic media, although it did not modify viability in either media. Ouabain also increased the percentage of swollen tails in the hypoosmotic medium at higher concentrations of the inhibitor. Amiloride increased the percentage of altered acrosomes in all media although its effect was less intense than that of ouabain. In isoosmotic media, total motility was decreased from about 80% in control to about 65% with 10(-4) M amiloride. Motile spermatozoa incubated with amiloride showed a clear decrease of mean velocity and mean linearity and increased frequency of head displacement. In the hyperosmotic medium, adding amiloride decreased the percentage of viability and altered tails at concentrations as low as 10(-6) to 10(-5) M. Our results indicate that the active mechanisms which control Na+ transport play a significant role in the maintenance of function in ram spermatozoa subjected to different osmotic environments. These mechanisms may be important in maintaining ram sperm function both "in vivo" and "in vitro".     

On the ionic mechanism underlying adrenergic-cholinergic antagonism in ventricular muscle

In atrial muscle, acetylcholine (ACh) decreases the slow inward current (Isi) and increases the time-independent outward K+ current. However, in ventricular muscle, ACh produces a marked negative inotropic effect only in the presence of positive inotropic agents that elevate cyclic adenosine monophosphate (AMP). A two-microelectrode voltage-clamp method was used on cultured reaggregates of cells from 16--20-d-old embryonic chick ventricles to determine the effects of ACh on Isi and outward current during beta-adrenergic stimulation. Only double penetrations displaying low-resistance coupling were voltage-clamped. Cultured reaggregates are advantageous because their small size (50-- 250 microns) permits better control of membrane potential and adequate space clamp. Tetrodotoxin (10(-6) M) and a holding potential of --50 to --40 mV were used to eliminate the fast Na+ current. Depolarizing voltage steps above --40 mV caused a slow inward current to flow that was sensitive to changes in [Ca]o and was depressed by verapamil (10(- 6) M). Maximal Isi was obtained at --10 mV and the reversal potential was about +25 mV. Isoproterenol (10(-6) M) increased Isi at all clamp potentials. Subsequent addition of ACh (10(-6) M) rapidly reduced Isi to control values (before isoproterenol) without a significant effect on the net outward current measured at 300 ms. The effects of ACh were reversed by muscarinic blockade with atropine (5 X 10(-6) M). We conclude that the anti-adrenergic effects of ACh in ventricular muscle are mediated by a reduction in Ca2+ influx during excitation.     

乙酰胆碱对蟾蜍背根神经节神经元膜电位的影响及离子机制

在离体灌流的蟾蜍背根神经节(DRG)标本上,用微电极进行胞内记录。在73个神经元中,依神经纤维的传导速度将神经元分为 A 型及 C 型,其中 A 型细胞67个,C 型6个,静息膜电位为-67.5±1.3mV((?)±SE)。当加4×10~(-4)—6×10~(-4)mol/L 乙酰胆碱(ACh),可观察到如下四种膜电位变化:1.超极化:幅值9.1±3.0mV((?)±SE,n=23);(2)去极化:幅值12.9±2.2mV((?)+SE,n=20);(3)双相反应(n=24):先超极化,后去极化,超极化幅值8.0±2.4mV((?)+SE),去极化幅值10.9±3.1mV((?)±SE);(4)无反应(n=6)。用阿托品(1.3×10~(-5)mol/L,n=23),或同时应用筒箭毒与六甲双铵(浓度均为1.4×10~(-5)mol/L,n=8)灌流,能分别阻断 ACh 引起的膜的超极化或去极化。ACh 引起超极化反应时膜电导平均增加13.8%,翻转电位值大约-96mV。四乙铵(TEA,20mmol/L)能使 ACh 的去极化幅值增加48.2±3.2%((?)±SE,n=6),超极化幅值减小79.4±4.3%((?)±SE,n=8)。MnCl_2(4mmol/L)使 ACh 的去极化及超极化幅值分别减小54.2±7.2%((?)±SE,n=5)及69.2±6.4%((?)±SE,n=14)。以上结果提示:ACh 引起的 DRG 神经细胞膜去极化反应由 N 型乙酰胆碱受体介导,而超极化反应由 Μ 型乙酰胆碱受体介导,前者可能包含了多种离子电导的改变,后者则可能与钾电导增加有关。     

Intracellular Na+ and K+ activities and membrane conductances in the collecting tubule of Amphiuma

Membrane potentials and conductances, and intracellular ionic activities were studied in isolated perfused collecting tubules of K+-adapted Amphiuma. Intracellular Na+ (aNai) and K+ (aKi) activities were measured, using liquid ion-exchanger double-barreled microelectrodes. Apical and basolateral membrane conductances were estimated by cable analysis. The effects of inhibition of the apical conductance by amiloride (10(-5) M) and of inhibition of the basolateral Na-K pump by either a low K+ (0.1 mM) bath or by ouabain (10(-4) M) were studied. Under control conditions, aNai was 8.4 +/- 1.9 mM and aKi 56 +/- 3 mM. With luminal amiloride, aNai decreased to 2.2 +/- 0.4 mM and aKi increased to 66 +/- 3 mM. Ouabain produced an increase of aNai to 44 +/- 4 mM, and a decrease of aKi to 22 +/- 6, and similar changes were observed when the tubule was exposed to a low K+ bath solution. During pump inhibition, there was a progressive decrease of the K+-selective basolateral membrane conductance and of the Na+ permeability of the apical membrane. A similar inhibition of both membrane conductances was observed after pump inhibition by low K+ solution. Upon reintroduction of K+, a basolateral membrane hyperpolarization of -23 +/- 4 mV was observed, indicating an immediate reactivation of the electrogenic Na-K pump. However, the recovery of the membrane conductances occurred over a slower time course. These data imply that both membrane conductances are regulated according to the intracellular ionic composition, but that the basolateral K+ conductance is not directly linked to the pump activity.     

Electrophysiological effects of basolateral [Na+] in Necturus gallbladder epithelium

In Necturus gallbladder epithelium, lowering serosal [Na+] ([Na+]s) reversibly hyperpolarized the basolateral cell membrane voltage (Vcs) and reduced the fractional resistance of the apical membrane (fRa). Previous results have suggested that there is no sizable basolateral Na+ conductance and that there are apical Ca(2+)-activated K+ channels. Here, we studied the mechanisms of the electrophysiological effects of lowering [Na+]s, in particular the possibility that an elevation in intracellular free [Ca2+] hyperpolarizes Vcs by increasing gK+. When [Na+]s was reduced from 100.5 to 10.5 mM (tetramethylammonium substitution), Vcs hyperpolarized from -68 +/- 2 to a peak value of -82 +/- 2 mV (P less than 0.001), and fRa decreased from 0.84 +/- 0.02 to 0.62 +/- 0.02 (P less than 0.001). Addition of 5 mM tetraethylammonium (TEA+) to the mucosal solution reduced both the hyperpolarization of Vcs and the change in fRa, whereas serosal addition of TEA+ had no effect. Ouabain (10(-4) M, serosal side) produced a small depolarization of Vcs and reduced the hyperpolarization upon lowering [Na+]s, without affecting the decrease in fRa. The effects of mucosal TEA+ and serosal ouabain were additive. Neither amiloride (10(-5) or 10(-3) M) nor tetrodotoxin (10(-6) M) had any effects on Vcs or fRa or on their responses to lowering [Na+]s, suggesting that basolateral Na+ channels do not contribute to the control membrane voltage or to the hyperpolarization upon lowering [Na+]s. The basolateral membrane depolarization upon elevating [K+]s was increased transiently during the hyperpolarization of Vcs upon lowering [Na+]s. Since cable analysis experiments show that basolateral membrane resistance increased, a decrease in basolateral Cl- conductance (gCl-) is the main cause of the increased K+ selectivity. Lowering [Na+]s increases intracellular free [Ca2+], which may be responsible for the increase in the apical membrane TEA(+)-sensitive gK+. We conclude that the decrease in fRa by lowering [Na+]s is mainly caused by an increase in intracellular free [Ca2+], which activates TEA(+)-sensitive maxi K+ channels at the apical membrane and decreases apical membrane resistance. The hyperpolarization of Vcs is due to increase in: (a) apical membrane gK+, (b) the contribution of the Na+ pump to Vcs, (c) basolateral membrane K+ selectivity (decreased gCl-), and (d) intraepithelial current flow brought about by a paracellular diffusion potential.     

Voltage clamp study of fast excitatory synaptic currents in bullfrog sympathetic ganglion cells

Excitatory postsynaptic currents (EPSCs) have been studied in voltage- clamped bullfrog sympathetic ganglion B cells. The EPSC was small, rose to a peak within 1-3 ms, and then decayed exponentially over most of its time-course. For 36 cells at --50 mV (21-23 degrees C), peak EPSC size was --6.5 +/- 3.5 nA (mean +/- SD), and the mean decay time constant tau was 5.3 +/- 0.9 ms. tau showed a small negative voltage dependence, which appeared independent of temperature, over the range -- 90 to --30 mV; the coefficient of voltage dependence was --0.0039 +/- 0.0014 mV-1 (n = 29). The peak current-voltage relationship was linear between --120 and --30 mV but often deviated from linearity at more positive potentials. The reversal potential determined by interpolation was approximately --5 mV. EPSC decay tau had a Q10 = 3. The commonly used cholinesterase inhibitors, neostigmine and physostigmine, exhibited complex actions at the ganglia. Neostigmine (1 X 10(-5)M) produced a time-dependent slowing of EPSC decay without consistent change in EPSC size. In addition, the decay phase often deviated from a single exponential function, although it retained its negative voltage dependence. With 1 x 10(-6) M physostigmine, EPSC decay was slowed by the decay phase remained exponential. At higher concentrations of physostigmine, EPSC decay was markedly prolonged and was composed of at least two decay components. High concentrations of atropine (10(-5) to 10(-4) M) produced complex alterations in EPSC decay, creating two or more exponential components; one decay component was faster and the other was slower than that observed in untreated cells. These results suggest that the time-course of ganglionic EPSC decay is primarily determined by the kinetics of the receptor-channel complex rather than hydrolysis or diffusion of transmitter away from the postsynaptic receptors.     

Effects of ouabain on the response to osmotic changes in dog and boar spermatozoa

Although there is much information on the response of spermatozoa from different species to osmotic changes, little has been reported about the mechanism/s by which spermatozoa react to similar changes in the osmotic pressure of the medium. In this study we examine the effect of inhibition of Na (+)K (+), ouabain-sensitive ATP-ase on the response of canine and porcine spermatozoa when they are incubated in hypoosmotic and hyperosmotic media. The presence of ouabain slightly decreased the percentages of total and progressive motility, and increased the percentages of altered acrosomes (from 13.0 +/- 0.3% to 17.2 +/- 0.4% in the presence of 10(-4) M ouabain) and, specially, swollen tails (from 0.6 +/- 0.1% to 5.9 +/- 0.2% in the presence of 10(-4) M of ouabain) in fresh dog semen, although it did not affect these parameters in boar semen samples. Moreover, ouabain increased the percentage of both altered acrosomes and swollen tails in canine spermatozoa incubated in 100 mOsm and in 900 mOsm media at concentrations higher than 10(-5) M and 10(-7) M, respectively. The percentage of viability of canine spermatozoa was not modified by ouabain after incubation in 100, 300 or 900 mOsm media. Furthermore, ouabain did not significantly affect boar spermatozoa incubated in 100, 300 or 900 mOsm media. Although ouabain induced a significant decrease in L-lactate production in canine spermatozoa in an isoosmotic medium (from 4.7 +/- 0.4 micromol mg protein x 60 min to 2.6 +/- 0.3 micromol mg protein x 60 min in the presence of 10(-4) M ouabain), there was no significant effect on L-lactate production in boar spermatozoa. These results indicate that while dog spermatozoa acted against changes in the osmotic pressure by a mechanism(s) related to Na (+)K (+), ouabain-sensitive ATP-ase, boar spermatozoa reacted to some mechanism(s) not related to ionic pumps.     

Correlation of the effect of Ca+2 on Na+ and K+ permeability and membrane potential of Ehrlich ascites tumor cells

The effect of Ca+2 on the transport and intracellular distribution of Na+ and K+ in Ehrlich ascites tumor cells was investigated in an effort to establish the mechanism of Ca+2-induced hyperpolarization of the cell membrane. Inclusion of Ca+2 (2 mM) in the incubation medium leads to reduced cytoplasmic concentrations of Na+, K+ and Cl- in steady cells. In cells inhibited by ouabain, Ca+2 causes a 41% decrease in the rate of net K+ loss, but is without effect on the rate of net Na+ accumulation. Net K+ flux is reduced by 50%, while net Na+ flux is unchanged in the transport-inhibited cells. The membrane potential of cells in Ca+2-free medium (-13.9 +/- 0.8 mV) is unaffected by the addition of ouabain. However, the potential of cells in Ca+2-containing medium (-23.3 +/- 1.2 mV) declines in one hour after the addition of ouabain to values comparable to those of control cells (-15.2 +/- 0.7 mV). The results of these experiments are consistent with the postulation that Ca+2 exerts two effects on Na+ and K+ transport. First, Ca+2 reduces the membrane permeability to K+ by 25%. Second, Ca+2 alters the coupling of the Na/K active transport mechanism leading to an electrogenic hyperpolarization of the membrane.     

An electrophysiological study of parthenogenetic activation in mammalian oocytes

Using conventional electrophysiological techniques, we have investigated the electrical responses of mouse and hamster oocytes in metaphase of the second meiotic division to agents which induce parthenogenetic activation. Oocytes from MF1 mice responded to 8.7% ethanol and to 0.3% benzyl alcohol by a depolarization (sometimes preceded by a brief hyperpolarization). The response to ethanol did not "desensitize," and the membrane potential recovered completely when the exposure to ethanol was interrupted. The response was accompanied by a decrease in membrane input resistance (Rin) and had an equilibrium potential of about +5 mV in standard medium and of -10mV in Na-free medium. The oocytes responded to A23187 and to La3+ by an increased Rin, and usually lysed during or after treatment. Multiphasic responses were elicited by ethanol and by Ca-ionophore in metaphase II hamster oocytes; an early hyperpolarization accompanied by a decreased Rin was a common feature of the response to both activating agents. The early hyperpolarization was no longer elicited when the cells were exposed for a second time to ethanol or A23187. K+ and Cl- were the ions mainly involved in the hyperpolarizing potential elicited by A23187, and K+ (but not Cl-) was the ionic species mainly involved in ethanol response. The above responses were peculiar to metaphase II oocytes since mouse and hamster ovarian oocytes (in prophase I) and fertilized eggs either failed to respond to the activating agents, or responded by increasing Rin. The variety of electrical responses to parthenogenetic agents indicates that in mammalian oocytes parthenogenetic activation is not triggered by a "classical" activation potential.     

Mechanically induced electrical responses in murine mammary epithelial cells in primary culture

In mouse mammary epithelial cells in primary culture, mechanical stimulation of a cell induced in other cells within the same colony a short depolarization of less than 15 mV with a duration of 1-8 s and a subsequent, prominent hyperpolarization of 6 mV lasting 10-40 s. Epidermal growth factor induces a spontaneous hyperpolarizing response in cultured mammary cells, and in cells treated with EGF mechanical stimulation produced a greater hyperpolarization, while the amplitude of the depolarizing response was not affected. The amplitude of the mechanically induced hyperpolarization was markedly reduced by quinine and tetraethylammonium, blockers of the Ca2+ -dependent K+ channel. The results suggest that the Ca2+ -dependent K+ channel was involved in the hyperpolarization.     

去甲肾上腺素引起蟾蜍背根神经节神经细胞膜电位变化的离子机制

本文应用细胞内记录方法,对去甲肾上腺素(NA)引起蟾蜍背根神经节(DRG)神经细胞膜电位去极化或超极化反应时的膜电导及翻转电位值进行了测量,并观察了钾和钙离子通道阻断剂灌流DRG对NA引起膜电位反应的影响。当NA引起去极化反应时,15个细胞的膜电导减小32.6％。少数细胞膜电导开始增加,继而减小(n=4)。NA超极化反应时膜电导增加13.2％(n=8)。NA去极化反应的翻转电位值为-88.5±0.9mV((?)±SE,n=4),NA超极化反应在膜电位处于-89至-92mV时消失。 钾通道阻断剂四乙铵可使NA去极化幅值增加73.7±11.9％((?)±SE,n=7),并使NA超极化幅值减小40.5％(n=4)。细胞内注入氯化铯使苯肾上腺素去极化幅值增加34.5％(n=4)。钙通道阻断剂氯化锰使NA去极化及超极化反应分别减小50.5±9.9％((?)±SE,n=10)和89.5±4.9％((?)±SE,n=7)。结果提示,NA引起DRG神经细胞膜电位的去极化或超极化反应,可能与膜的钾及钙通道活动的改变有关。     

Evidence for the involvement of myoendothelial gap junctions in EDHF-mediated relaxation in the rat middle cerebral artery

The mechanisms underlying endothelium-dependent hyperpolarizing factor (EDHF) in the middle cerebral artery (MCA) remain largely unresolved. In particular, very little is known regarding the way in which the signal is transmitted from endothelium to smooth muscle. The present study tested the hypothesis that direct communication via myoendothelial gap junctions contributes to the EDHF response in the male rat MCA. EDHF-mediated dilations were elicited in rat MCAs by luminal application of ATP or UTP in the presence of Nomega-nitro-L-arginine methyl ester and indomethacin. Maximum dilation to luminal ATP (10(-4) M) was reduced significantly after incubation with a gap peptide cocktail (9 +/- 4%, n = 6) compared with a scrambled gap peptide cocktail (99 +/- 1%, n = 6, P < 0.05). A gap peptide cocktail had no effect on amplitude of endothelial cell hyperpolarization in response to 3 x 10(-5) M UTP (22 +/- 3 vs. 22 +/- 1 mV, n = 4), whereas smooth muscle cell hyperpolarization was significantly attenuated (17 +/- 1 vs. 6 +/- 1 mV, n = 4, P = 0.004). Connexin (Cx) 37 was localized to smooth muscle and Cx43 to endothelium, whereas Cx40 was found in endothelium and smooth muscle. Electron microscopy revealed the existence of frequent myoendothelial junctions. The total number of myoendothelial junctions per 5 microm of MCA sectioned was 2.5 +/- 0.5. Our results suggest that myoendothelial communication contributes to smooth muscle cell hyperpolarization and EDHF dilation in male rat MCA.     

Electrophysiological studies on the cultured cells obtained from transplantable pancreatic carcinoma in Syrian golden hamsters

A pancreatic ductal carcinoma was established as a transplantable tumor line in an inbred strain of Syrian golden hamsters. Intracellular recordings of membrane potentials and input resistance were made from cultured cells obtained from the transplanted tumors using indwelling glass microelectrode. The mean value of the resting membrane potential was -46.5 +/- 1.8 mV (S.E.) (n = 13), while the mean resting input resistance was 21.2 +/- 4.3 M omega (S.E.) (N = 13). Dibutyryl cyclic AMP (2 X 10(-3)M) caused a marked hyperpolarization of about 30 mV accompanied by a reduction of input resistance. The transplantable tumor and its cultured cell line developed in this study have demonstrated their effectiveness as a reliable experimental model for use in pancreatic cancer research.     

Hyperpolarization of mouse skeletal muscle plasma membrane induced by extracellular NADH

Extracellularly applied NADH, but not NAD or NADPH, increases the resting membrane potential from -74.1 to -76.6 mV in freshly isolated muscles in the presence of K+ in the incubation medium and from -64.6 to -72.9 mV in muscles equilibrated for 4-6 h in a K+-free solution. The NADH-induced hyperpolarization is blocked by pretreatment of muscles with ouabain, and the inhibitors of plasma membrane NADH dehydrogenase (adriamycin, azide, PCMB, atebrine, DIDS and bleomycin). The effect of NADH is accompanied by the disappearance of NADH from the incubation medium and by decreased membrane resistance. We conclude that NADH hyperpolarization is due to the enhancement of passive membrane permeability, apparently for K+, which might result from the conformational changes in the plasma membrane during the NADH dehydrogenase reaction. The possibility is discussed that NADH dehydrogenase mediates transport of K+ out from the cell using a pathway connected with the transmembrane Na+/K+ pump.     

Identification of two molecular forms of (Na+,K+)-ATPase in rat adipocytes. Relation to insulin stimulation of the enzyme

Two molecular forms of the (Na+,K+)-ATPase catalytic subunit have been identified in rat adipocyte plasma membranes using immunological techniques. The similarity between these two forms and those in brain (Sweadner, K. J. (1979) J. Biol. Chem. 254, 6060-6067) led us to use the same nomenclature: alpha and alpha(+). The K0.5 values of each form for ouabain (determined by inhibition of phosphorylation of the enzyme from [gamma-32P]ATP) were 3 X 10(-7)M for alpha(+) and 1 X 10(-5)M for alpha. These numbers correlate well with the K0.5 values for the two ouabain-inhibitable components of 86Rb+/K+ pumping in intact cells (1 X 10(-7) M and 4 X 10(-5)M). Quantitation of the Na+ pumps in plasma membranes demonstrated a total of 11.5 +/- 0.2 pmol/mg of membrane protein, of which 8.5 +/- 0.3 pmol/mg, or 75%, was alpha(+). Insulin stimulation of 86Rb+/K+ uptake in rat adipocytes was abolished by ouabain at a concentration sufficient to inhibit only alpha(+)(2-5 X 10(-6)M). Immunological techniques and ouabain inhibition of catalytic labeling of the enzyme from [gamma-32P]ATP demonstrated that alpha(+) was present in skeletal muscle membranes as well as in adipocyte membranes, but was absent from liver membranes. Since insulin stimulates increased Na+ pump activity in adipose and muscle tissue but not in liver, there is a correlation between hormonal regulation of (Na+,K+)-ATPase and the presence of alpha(+). We propose that alpha(+) is the hormonally-sensitive version of the enzyme.     

Multiple channels mediate calcium leakage in the A7r5 smooth muscle-derived cell line.

Ca2+ entry under resting conditions may be important for contraction of vascular smooth muscle, but little is known about the mechanisms involved. Ca2+ leakage was studied in the A7r5 smooth muscle-derived cell line by patch-clamp techniques. Two channels that could mediate calcium influx at resting membrane potentials were characterized. In 110 mM Ba2+, one channel had a slope conductance of 6.0 +/- 0.6 pS and an extrapolated reversal potential of +41 +/- 13 mV (mean +/- SD, n = 8). The current rectified strongly, with no detectable outward current, even at +90 mV. Channel gating was voltage independent. A second type of channel had a linear current-voltage relationship, a slope conductance of 17.0 +/- 3.2 pS, and a reversal potential of +7 +/- 4 mV (n = 9). The open probability increased e-fold per 44 +/- 10 mV depolarization (n = 5). Both channels were also observed in 110 mM Ca2+. Noise analysis of whole-cell currents indicates that approximately 100 6-pS channels and 30 17-pS channels are open per cell. These 6-pS and 17-pS channels may contribute to resting calcium entry in vascular smooth muscle cells.     

Norepinephrine-stimulated vascular prostacyclin synthesis. Receptor-dependent calcium channels control prostaglandin synthesis

Norepinephrine-stimulated prostacyclin synthesis was studied in rat aortic rings by measuring 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) by radioimmunoassay. Norepinephrine (10(-6) M) results in a 10- to 20-fold increase in 6-keto-PGF1 alpha synthesis by rat aortic rings (54 +/- 11 to 437 +/- 260 pg X mg wet weight-1 X 20 min-1). The maximal stimulation of 6-keto-PGF1 alpha synthesis was observed with a norepinephrine concentration of 10(-5) M at a mean effective concentration (EC50) of 9.5 +/- 3.2 X 10(-7) M which is similar to the contractile response (Emax = 10(-5) M, EC50 = 6.5 +/- 1.8 X 10(-7) M). Potassium chloride (30 mM), although causing a similar maximal contractile response as 10(-6) M norepinephrine, did not increase 6-keto-PGF1 alpha synthesis. Norepinephrine-stimulated 6-keto-PGF1 alpha synthesis was dependent upon extracellular calcium. Norepinephrine stimulation in Ca2+-free medium did not lead to a significant increase in 6-keto-PGF1 alpha synthesis. However, on the introduction of Ca2+, 6-keto-PGF1 alpha synthesis was restored to its initial level. Phentolamine (10(-6) M) (an alpha-adrenergic antagonist) and trifluroperazine (2.5 X 10(-4) M) (a calmodulin inhibitor) completely inhibited norepinephrine-stimulated 6-keto-PGF1 alpha synthesis, whereas verapamil 3 X 10(-6) M (a calcium channel blocking drug) only partially inhibited synthesis (control, 74 +/- 12; norepinephrine, 437 +/- 260; norepinephrine + verapamil, 123 +/- 8 pg X mg wet weight-1 X 20 min-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for Ca2+ control of the transducer mechanism in crayfish stretch receptor.

Recording from the dendrite membrane indicated a resting potential of --51.6 mV, which was reduced by inhibition of the Na+/K+ pump. Voltage clamp at rest revealed a small inward current between --50 and --80 mV and a larger outward current at clamp potentials of --40 to plus 30 mV. Using ramp-changes of muscle tension as stimuli a time-variant tension-induced inward current (TIC) became apparent, the amplitude of which decreased towards larger depolarizing voltages until at plus 18 mV the current reversed the direction. The time course of the conductance changes corresponds to similar phases in the generator potential. The outward current only responded to fast reductions in tension, decreasing transiently. A contribution of the active Na+/K+ pump to the hyperpolarizing potential response is suggested by the effects of K-removal or Na-substitution by Li+. In Na-free choline chloride media the generator potential and the TIC was depressed by 70-85%. Additional removal of Ca2+ abolished the TIC. In contrast, lowering the Ca2+ level in presence of Na+ decreased the membrane resistance and markedly enhanced the TIC (maximally eightfold at 10(-5) M Ca2+) while 75-150 mM Ca2+ or intracellular application of a Ca-ionophore had the reverse effect.     

Effects of intracellular K+ and Rb+ on gating of embryonic rat telencephalon Ca(2+)-activated K+ channels.

We have investigated the effects of intracellular K+ and Rb+ on single-channel currents recorded from the large-conductance Ca(2+)-activated K+ (BK) channel of the embryonic rat telencephalon using the inside-out patch-clamp technique. Our novel observation concerns the effects of these ions on rapid flickering of channel openings. Specifically, flicker gating was voltage dependent, i.e., it was reduced by depolarization in the -60 to -10 mV range with equimolar concentrations of K+ ions (150 Ko+/150 Ki+). Removal of Ki+ resulted in significant flickering at all potentials in this voltage range. In other words, the voltage dependence of flicker gating was effectively eliminated by the removal of Ki+. This suggests that a K+ ion entering the channel from the intracellular medium binds, in a voltage-dependent manner, at a site that locks the flicker gate in its open position. No effects of changes in Ki+ were observed on the primary, voltage-dependent gate of the channel. The change in flickering did not cause a change in the mean burst duration, which indicates that the primary gate is stochastically independent of the flicker gate. Intracellular Rb+ can substitute for--and is even more effective than--Ki+ with regard to suppression of flickering. Substitution of Rbi+ for Ki+ also increased the mean burst duration for V > or = -30 mV. Both effects of Rbi+ were removed by membrane hyperpolarization.