Angiotensin II promotes selective uptake of high density lipoprotein cholesterol esters in bovine adrenal glomerulosa and human adrenocortical carcinoma cells through induction of scavenger receptor class B type I.

Angiotensin II is one of the main physiological regulators of aldosterone biosynthesis in the zona glomerulosa of the adrenal cortex. The hormone stimulates intracellular cholesterol mobilization to the mitochondrion for steroid biosynthesis. Here we have examined whether angiotensin II also modulates exogenous lipoprotein cholesterol ester supply to the steroidogenic machinery and whether this control is exerted on the selective transport of high density lipoprotein-derived cholesterol ester to intracellular lipid droplets through the scavenger receptor class B type I. In bovine adrenal glomerulosa and human NCI H295R adrenocortical carcinoma cells, high density lipoprotein stimulated steroid production. Angiotensin II pretreatment for 24 h potentiated this response. Fluorescence microscopy of cellular uptake of reconstituted high density lipoprotein containing a fluorescent cholesterol ester revealed an initial, time-dependent narrow labeling of the cell membrane followed by an intense accumulation of the fluorescent cholesterol ester within lipid droplets. At all time points, labeling was more pronounced in cells that had been treated for 24 h with angiotensin II. Fluorescence incorporation into cells was prevented by a monoclonal antibody directed against apolipoprotein A-I. Upon quantitative fluorometric determination, cholesterol ester uptake in angiotensin II-treated bovine cells was increased to 175 +/- 15% of controls after 2 h and to 260 +/- 10% after 4 h of exposure to fluorescent high density lipoprotein. The amount of scavenger receptor class B type I protein detected in cells treated with angiotensin II for 24 h reached 203 +/- 12% of that measured in control cells (n = 3, P < 0.01). In contrast, low density lipoprotein receptors were only minimally affected by angiotensin II treatment. This increase in scavenger receptor class B type I protein was associated with a 3-fold induction of scavenger receptor class B type I mRNA, which could be prevented by actinomycin D but not by cycloheximide. Similar results were obtained in the human adenocarcinoma cell line H295R. These observations show that angiotensin II regulates the scavenger receptor class B type I-mediated selective transport of lipoprotein cholesterol ester across the cell membrane as a major source of precursor for mineralocorticoid biosynthesis in both human and bovine adrenal cells.

Functional specialization of calreticulin domains.

Calreticulin is a Ca2+-binding chaperone in the endoplasmic reticulum (ER), and calreticulin gene knockout is embryonic lethal. Here, we used calreticulin-deficient mouse embryonic fibroblasts to examine the function of calreticulin as a regulator of Ca2+ homeostasis. In cells without calreticulin, the ER has a lower capacity for Ca2+ storage, although the free ER luminal Ca2+ concentration is unchanged. Calreticulin-deficient cells show inhibited Ca2+ release in response to bradykinin, yet they release Ca2+ upon direct activation with the inositol 1,4,5-trisphosphate (InsP3). These cells fail to produce a measurable level of InsP3 upon stimulation with bradykinin, likely because the binding of bradykinin to its cell surface receptor is impaired. Bradykinin binding and bradykinin-induced Ca2+ release are both restored by expression of full-length calreticulin and the N + P domain of the protein. Expression of the P + C domain of calreticulin does not affect bradykinin-induced Ca2+ release but restores the ER Ca2+ storage capacity. Our results indicate that calreticulin may play a role in folding of the bradykinin receptor, which affects its ability to initiate InsP3-dependent Ca2+ release in calreticulin-deficient cells. We concluded that the C domain of calreticulin plays a role in Ca2+ storage and that the N domain may participate in its chaperone functions.

Heme histidine ligands within gp91(phox) modulate proton conduction by the phagocyte NADPH oxidase.

The membrane subunit of the phagocyte NADPH oxidase, gp91(phox), possesses a H(+) channel motif formed by membrane-spanning histidines postulated to coordinate the two heme groups forming the redox center of the flavocytochrome. To study the role of heme-binding histidines on proton conduction, we stably expressed the gp91(phox) cytochrome in human embryonic kidney 293 cells and measured proton currents with the patch clamp technique. Similar to its shorter homologue, NADPH oxidase homologue 1, which is predicted not to bind heme, gp91(phox) generated voltage-activated, pH-dependent, H(+)-selective currents that were reversibly blocked by Zn(2+). The gp91(phox) currents, however, activated faster, deactivated more slowly, and were markedly affected by the inhibition of heme synthesis. Upon heme removal, the currents had larger amplitude, activated faster and at lower voltages, and became sensitive to the histidine reagent diethylpyrocarbonate. Mutation of the His-115 residue to leucine abolished both the gp91(phox) characteristic 558-nm absorbance peak and voltage-activated currents, indicating that His-115 is involved in both heme ligation and proton conduction. These results indicate that the gp91(phox) proton channel is activated upon release of heme from its His-115 ligand. During activation of the oxidase complex, changes in heme coordination within the cytochrome might increase the mobility of histidine ligands, thereby coupling electron and proton transport.

Mitochondria recycle Ca(2+) to the endoplasmic reticulum and prevent the depletion of neighboring endoplasmic reticulum regions.

To study Ca(2+) fluxes between mitochondria and the endoplasmic reticulum (ER), we used "cameleon" indicators targeted to the cytosol, the ER lumen, and the mitochondrial matrix. High affinity mitochondrial probes saturated in approximately 20% of mitochondria during histamine stimulation of HeLa cells, whereas a low affinity probe reported averaged peak values of 106 +/- 5 microm, indicating that Ca(2+) transients reach high levels in a fraction of mitochondria. In concurrent ER measurements, [Ca(2+)](ER) averaged 371 +/- 21 microm at rest and decreased to 133 +/- 14 microm and 59 +/- 5 microm upon stimulation with histamine and thapsigargin, respectively, indicating that substantial ER refilling occur during agonist stimulation. A larger ER depletion was observed when mitochondrial Ca(2+) uptake was prevented by oligomycin and rotenone or when Ca(2+) efflux from mitochondria was blocked by CGP 37157, indicating that some of the Ca(2+) taken up by mitochondria is re-used for ER refilling. Accordingly, ER regions close to mitochondria released less Ca(2+) than ER regions lacking mitochondria. The ER heterogeneity was abolished by thapsigargin, oligomycin/rotenone, or CGP 37157, indicating that mitochondrial Ca(2+) uptake locally modulate ER refilling. These observations indicate that some mitochondria are very close to the sites of Ca(2+) release and recycle a substantial portion of the captured Ca(2+) back to vicinal ER domains. The distance between the two organelles thus determines both the amplitude of mitochondrial Ca(2+) signals and the filling state of neighboring ER regions.

Inositol 1,4,5-trisphosphate directs Ca(2+) flow between mitochondria and the Endoplasmic/Sarcoplasmic reticulum: a role in regulating cardiac autonomic Ca(2+) spiking.

The signaling role of the Ca(2+) releaser inositol 1,4, 5-trisphosphate (IP(3)) has been associated with diverse cell functions. Yet, the physiological significance of IP(3) in tissues that feature a ryanodine-sensitive sarcoplasmic reticulum has remained elusive. IP(3) generated by photolysis of caged IP(3) or by purinergic activation of phospholipase Cgamma slowed down or abolished autonomic Ca(2+) spiking in neonatal rat cardiomyocytes. Microinjection of heparin, blocking dominant-negative fusion protein, or anti-phospholipase Cgamma antibody prevented the IP(3)-mediated purinergic effect. IP(3) triggered a ryanodine- and caffeine-insensitive Ca(2+) release restricted to the perinuclear region. In cells loaded with Rhod2 or expressing a mitochondria-targeted cameleon and TMRM to monitor mitochondrial Ca(2+) and potential, IP(3) induced transient Ca(2+) loading and depolarization of the organelles. These mitochondrial changes were associated with Ca(2+) depletion of the sarcoplasmic reticulum and preceded the arrest of cellular Ca(2+) spiking. Thus, IP(3) acting within a restricted cellular region regulates the dynamic of calcium flow between mitochondria and the endoplasmic/sarcoplasmic reticulum. We have thus uncovered a novel role for IP(3) in excitable cells, the regulation of cardiac autonomic activity.

Bcl-2 decreases the free Ca2+ concentration within the endoplasmic reticulum.

The antiapoptotic protein Bcl-2 localizes not only to mitochondria but also to the endoplasmic reticulum (ER). However, the function of Bcl-2 at the level of the ER is poorly understood. In this study, we have investigated the effects of Bcl-2 expression on Ca(2+) storage and release by the ER. The expression of Bcl-2 decreased the amount of Ca(2+) that could be released from intracellular stores, regardless of the mode of store depletion, the cell type, or the species from which Bcl-2 was derived. Bcl-2 also decreased cellular Ca(2+) store content in the presence of mitochondrial inhibitors, suggesting that its effects were not mediated through mitochondrial Ca(2+) uptake. Direct measurements with ER-targeted Ca(2+)-sensitive fluorescent "cameleon" proteins revealed that Bcl-2 decreased the free Ca(2+) concentration within the lumen of the ER, [Ca(2+)](ER). Analysis of the kinetics of Ca(2+) store depletion in response to the Ca(2+)-ATPase inhibitor thapsigargin revealed that Bcl-2 increased the permeability of the ER membrane. These results suggest that Bcl-2 decreases the free Ca(2+) concentration within the ER lumen by increasing the Ca(2+) permeability of the ER membrane. The increased ER Ca(2+) permeability conferred by Bcl-2 would be compatible with an ion channel function of Bcl-2 at the level of the ER membrane.

A mammalian H+ channel generated through alternative splicing of the NADPH oxidase homolog NOH-1.

Voltage-gated proton (H+) channels are found in many human and animal tissues and play an important role in cellular defense against acidic stress. However, a molecular identification of these unique ion conductances has so far not been achieved. A 191-amino acid protein is described that, upon heterologous expression, has properties indistinguishable from those of native H+ channels. This protein is generated through alternative splicing of messenger RNA derived from the gene NOH-1 (NADPH oxidase homolog 1, where NADPH is the reduced form of nicotinamide adenine dinucleotide phosphate).

L-type and Ca2+ release channel-dependent hierarchical Ca2+ signalling in rat portal vein myocytes.

Ca2+ signalling events and whole-cell Ca2+ currents were analyzed in single myocytes from rat portal vein by using a laser scanning confocal microscope combined with the patch-clamp technique. In myocytes in which the intracellular Ca2+ store was depleted or Ca2+ release channels were blocked by 10 microM ryanodine, inward Ca2+ currents induced slow and sustained elevations of [Ca2+]i. These Ca2+ responses were suppressed by 1 microM oxodipine and by depolarizations to +120 mV, a potential close to the reversal potential for Ca2+ ions, suggesting that they reflected Ca2+ influx through L-type Ca2+ channels. With functioning intracellular Ca2+ stores, flash photolysis of caged Ca2+ gave rise to a small increase in [Ca2+]i with superimposed Ca2+ sparks, reflecting the opening of clustered Ca2+ release channels. Brief Ca2+ currents in the voltage range from -30 to +10 mV triggered Ca2+ sparks or macrosparks that did not propagate in the entire line-scan image. Increasing the duration of Ca2+ current for 100 ms or more allowed the trigger of propagating Ca2+ waves which originated from the same initiation sites as the caffeine-activated response. Both Ca2+ sparks and initiation sites of Ca2+ waves activated by Ca2+ currents were observed in the vicinity of areas that excluded the Ca2+ probes, reflecting infoldings of the plasma membrane close to the sarcoplasmic reticulum, as revealed by fluorescent markers. The hierarchy of Ca2+ signalling events, from submicroscopic fundamental events to elementary events (sparks) and propagated waves, provides an integrated mechanism to regulate vascular tone.

Effect of a 14-day hindlimb suspension on cytosolic Ca2+ concentration in rat portal vein myocytes.

Effects of a 14-day hindlimb suspension were examined on increases in cytosolic Ca2+ concentration ([Ca2+]i) evoked by vasoactive compounds and on Ca2+ channels in rat portal vein myocytes. The maximal increases in [Ca2+]i elicited by caffeine, norepinephrine, and angiotensin II were reduced by 30-50% in suspended rats, and complete recovery was obtained 4 days after suspension removal. In contrast, voltage-gated Ca2+ channels were unaffected by hindlimb suspension. Using both confocal microscopy and the patch-clamp technique, we measured local increases in [Ca2+]i which corresponded to activation of a small number of ryanodine-sensitive Ca(2+)-release channels (Ca2+ sparks) and D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-gated Ca2+ channels. After hindlimb suspension, these local Ca2+ events, as well as the Ca2+ sensitivity of ryanodine-sensitive Ca2+ release channels, remained unchanged. In contrast, the propagated Ca2+ responses (Ca2+ waves) were significantly reduced in parallel with a noticeable inhibition of [3H]ryanodine binding to vascular membranes. Taken together, these results suggest that inhibition of the vasoconstrictor-induced increases in [Ca2+]i during long-term suspension may be related to a reduction of the number of functional ryanodine-sensitive and Ins(1,4,5)P3-gated channels in the sarcoplasmic reticulum of rat portal vein myocytes.

Ca2+ sparks and Ca2+ waves activate different Ca(2+)-dependent ion channels in single myocytes from rat portal vein.

Ca2+ release from intracellular stores was examined with the use of a confocal microscope in single, voltage-clamped myocytes from rat portal vein loaded with both Fluo-3 and Fura-red. Spontaneous local increases in [Ca2+]i from the sarcoplasmic reticulum, termed Ca2+ sparks, were observed in about 30% of the quiescent cells tested. Ca2+ sparks could be evoked by low concentrations of caffeine (1 mM) or ryanodine (1 microM). Both spontaneous and caffeine-evoked Ca2+ sparks were insensitive to blockers of voltage-dependent Ca2+ channels. Caffeine (10 mM) triggered propagating Ca2+ waves of large amplitude which started from the same site than spontaneous Ca2+ sparks in 73% of the cells, as expected if Ca2+ sparks were the elementary events that could account for the initiation of Ca2+ waves. Spontaneous Ca2+ sparks activated both Ca(2+)-dependent K+ and non-selective cation currents, whereas Ca2+ waves were able to evoke Ca(2+)-dependent chloride current. These results suggest that both inward cation current and outward K+ current activated by Ca2+ sparks may exert a key role in controlling the basal activity of vascular myocytes.

Activation of calcium sparks by angiotensin II in vascular myocytes.

Contraction in smooth muscle is triggered by an increase in cytoplasmic free calcium ([Ca2+]i) which depends on both Ca2+ influx through L-type Ca2+ channels and Ca2+ release from the sarcoplasmic reticulum (SR). Two mechanisms have been shown to be involved in SR Ca2+ release, one is stimulated by Ca2+ and involved ryanodine-sensitive Ca2+-release channels; the other is stimulated by an increase in inositol 1,4,5-trisphosphate (InsP3) generation induced by various mediators and involved InsP3-sensitive Ca2+ release channels. Here, we examined the effects of angiotensin II on [Ca2+]i in single rat portal vein myocytes using both the whole cell patch-clamp method and a laser scanning confocal microscope. Elementary Ca2+ release events (Ca2+ sparks) were obtained spontaneously or in response to L-type Ca2+ channel current activation, and resulted from activation of ryanodine-sensitive Ca2+-release channels in the SR. We show that angiotensin AT1 receptors stimulate Ca2+ sparks through activation of L-type Ca2+ channels without involving InsP3-induced Ca2+ release. This novel transduction pathway may be a common mechanism for vasoconstrictors which do not stimulate generation of chemical second messengers.

Beta adrenergic receptor activation attenuates the generation of inositol phosphates in the pregnant rat myometrium. Correlation with inhibition of Ca++ influx, a cAMP-independent mechanism.

In the pregnant rat myometrium, an averaged 30% of inositol phosphate accumulation induced by carbachol and oxytocin was inhibited by oxodipine indicating that a part of receptor-mediated generation of inositol phosphates depended on Ca++ influx through voltage-gated Ca++ channels. In fura-2-loaded cells, carbachol and oxytocin caused a two-phase [Ca++]i response, made up of a transient [Ca++]i peak of about 700 nM followed by a sustained phase of about 120 nM. Oxodipine reduced the [Ca++]i peak by 40% and the plateau phase by 50%, pointing to a contribution of Ca++ influx in both the [Ca++]i peak and sustained phase. Isoproterenol reduced inositol phosphate response to carbachol and oxytocin to an amount equivalent to that elicited by oxodipine. No additional reduction could be obtained in a combination of isoproterenol and oxodipine. Isoproterenol decreased by 40% the [Ca++]i peak and by 70% the [Ca++]i plateau phase. Differently from isoproterenol, forskolin did not affect inositol phosphate accumulation induced by oxytocin and failed to attenuate the [Ca++]i peak. The inhibitory effect of isoproterenol on both inositol phosphate accumulation and [Ca++]i increase induced by oxytocin was abolished by pertussis toxin. These data suggest that beta adrenergic receptor activation is linked via a cAMP-independent, pertussis toxin-sensitive process to an activation of K+ channels, as revealed by use of selective K+ channel antagonists, with the consequent closure of voltage-gated Ca++ channels, resulting in the inhibition of the Ca(++)-associated generation of inositol phosphates.

A Gi1-2-protein is required for alpha 2A-adrenoceptor-induced stimulation of voltage-dependent Ca2+ channels in rat portal vein myocytes.

In rat portal vein myocytes, alpha 2A-adrenoceptors activate voltage-dependent Ca2+ channels via a transduction pathway requiring protein kinase C activation mediated by a pertussis-toxin-sensitive G-protein. As revealed by the use of antibodies directed against the different alpha-subunits expressed in portal vein myocytes, we show that the clonidine-induced stimulation of voltage-dependent Ca2+ channels is mainly mediated by a Gi1-2-protein.

Electrophysiological and radioligand binding studies of elgodipine and derivatives in portal vein myocytes.

The effects of a novel dihydropyridine, elgodipine, and of three derivatives have been studied on the calcium channel currents of isolated cells from rat portal vein by the patch-clamp technique, and on specific (+)-[3H]isradipine binding to vascular membranes. Elgodipine inhibited both T- and L-type calcium channels in a concentration-dependent manner. Half-inhibitions of T- and L-type calcium channel current were obtained at concentrations of 32 and 2.3 nM, respectively. Currents activated repetitively were similarly inhibited than those after a rest period, indicating absence of use-dependent inhibition by elgodipine. When cells were held at depolarized membrane potentials at which T- or L-type calcium channels were inactivated, the inhibitory effects of elgodipine were enhanced on both calcium channel currents, indicating that the elgodipine-induced inhibition was voltage-dependent. The elgodipine concentration which blocked the inactivated calcium channels were 5 to 7 times lower than those which blocked the resting calcium channels. The inhibition constant for elgodipine obtained from the displacement of (+)-[3H]isradipine binding to the L-type calcium channels in vascular membranes was identical to the dissociation constant calculated from electrophysiological data on inactivated calcium channels. At concentrations that completely inhibited calcium channels, elgodipine had no effect on chloride and potassium channels, and did not interfere with the intracellular calcium stores.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxytocin mobilizes calcium from a unique heparin-sensitive and thapsigargin-sensitive store in single myometrial cells from pregnant rats.

Intracellular free Ca2+ concentration ([Ca2+]i) was monitored using the fluorescence from the dye Fura-2-AM in single myometrial cells from pregnant rats. Oxytocin and acetylcholine applied to the cell evoked an initial peak in [Ca2+]i followed by a smaller sustained rise which was rapidly terminated upon removal of acetylcholine or persisted after oxytocin removal. A Ca2+ channel blocker (oxodipine) and external Ca2+ removal decreased both the transient and sustained rises in [Ca2+]i suggesting that Ca2+ influx through L-type Ca2+ channels participated in the global Ca2+ response induced by oxytocin. However, the initial peak in [Ca2+]i produced by oxytocin was mainly due to Ca2+ store release: it was abolished by inclusion of heparin [which blocks inositol 1,4,5-trisphosphate (InsP3) receptors] in the pipette (whole-cell recording mode of patch-clamp) and external application of thapsigargin (which blocks sarcoplasmic reticulum Ca(2+)-ATPases). In contrast, the transient Ca2+ response induced by oxytocin was unaffected by ryanodine. Moreover, caffeine failed to induce a rise in [Ca2+]i but reduced the oxytocin-induced transient Ca2+ response. The later sustained rise in [Ca2+]i produced by oxytocin was due to the entry of Ca2+ into the cell as it was suppressed in external Ca(2+)-free solution. The Ca2+ entry pathway is permeable to Mn2+ ions, in contrast to that described in various vascular and visceral smooth muscle cells. Oxytocin-induced Ca2+ release is blocked by the oxytocin antagonist d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT. The prolonged increase in [Ca2+]i after oxytocin removal is rapidly terminated by addition of the oxytocin antagonist suggesting that oxytocin dissociation from its receptor is very slow.(ABSTRACT TRUNCATED AT 250 WORDS)

Chloride and monovalent ion-selective cation currents activated by oxytocin in pregnant rat myometrial cells.

OBJECTIVE: The purpose of our study was to characterize the membrane mechanisms responsible for oxytocin-induced depolarization in single cells from pregnant rat myometrium. STUDY DESIGN: Membrane currents were recorded with the whole-cell mode of the standard patch-clamp technique. Intracellular calcium concentration was monitored with the fluorescence from Fura 2 added to the pipette solution. RESULTS: We found that oxytocin predominantly activates potassium, chloride, and cation conductances. Chloride and cation currents were evoked by an increase in the intracellular calcium concentration dependent on calcium release from the heparin-sensitive intracellular stores. Chloride and cation current showed different calcium dependences so that they could be activated separately. CONCLUSION: Stimulation of oxytocin receptors induces opening of calcium-activated chloride and cation channels, leading to depolarization of the myometrial cells. This depolarization opens, in turn, voltage-dependent calcium channels.

Activation of alpha-1A adrenoceptors mobilizes calcium from the intracellular stores in myocytes from rat portal vein.

Intracellular free Ca++ concentration ([Ca++]i) was monitored using the fluorescence from the dye fura-2-acetoxymethylester in single myocytes from rat portal vein. In the presence of oxodipine (a L-type Ca++ channel inhibitor), norepinephrine (10 microM) evoked transient increases in [Ca++]i which were related to release of Ca++ from intracellular stores. The alpha-1 adrenoceptors mediating intracellular Ca++ release and inositol phosphate accumulation were identified by using subtype-selective agonists and antagonists. Pretreatment with chloroethylclonidine had little effect on the norepinephrine-induced increase in [Ca++]i and inositol phosphate accumulation. In contrast, prazosin, 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane and alpha-ethyl-3,4,5-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)ethyl)-amino )- propyl)benzeneacetonitrile fumarate produced a concentration-dependent inhibition of both intracellular Ca++ release and inositol phosphate accumulation. The rank of potency was prazosin > 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane > alpha-ethyl-3,4,5-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)ethyl)-amino - propyl) benzeneacetonitrile fumarate. Methoxamine was as effective as norepinephrine but was less potent as shown by the rightward shift of the concentration-response curves. These results indicate that myocytes from rat portal vein express alpha-1A adrenoceptors whose activation stimulates phosphoinositide turnover and release of Ca++ from intracellular stores. The alpha-1A adrenoceptor stimulation of [Ca++]i and subsequent activation of Ca(++)-activated Cl- current was insensitive to intracellular applications of pertussis toxin, but concentration-dependently blocked by intracellular dialysis with a pipette solution containing anti-alpha q/alpha 11 antibody (whole cell recording mode).(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of dihydropyridines on calcium channels in isolated smooth muscle cells from rat vena cava.

1. Whole-cell patch-clamp method was applied to single smooth muscle cells freshly isolated from the rat inferior vena cava. 2. Depolarizing pulses, applied from a holding potential of -90 mV, activated both Na+ and Ca2+ channels. The fast Na+ current was inhibited by nanomolar concentrations of tetrodotoxin (TTX). The slow Ba2+ current (measured in 5 mM Ba2+ solution) was inhibited by Cd2+ and modulated by dihydropyridine derivatives. When the cells were held at a holding potential of -80 mV, racemic Bay K 8644 increased the Ba2+ current (ED50 = 10 nM) while racemic isradipine inhibited the current (IC50 = 21 nM). 3. The voltage-dependency of isradipine blockade was assessed by determining the steady-state availability of the Ca2+ channels. From the shift of the inactivation curve in the presence of isradipine, we calculated a dissociation constant of 1.11 nM for inactivated Ca2+ channels. Scatchard plots of the specific binding of (+)-[3H]-isradipine obtained in intact strips incubated in 5.6 mM or 135 mM K+ solutions confirmed the voltage-dependency of isradipine binding. 4. Specific binding of (+)-[3H]-isradipine was completely displaced by unlabelled (+/-)-isradipine, with an IC50 of 15.1 nM. This value is similar to the IC50 for inhibition of the Ba2+ current (21 nM) in cells maintained at a holding potential of -80 mV. 5. Bay K 8644 had no effects on the Ba2+ current kinetics during a depolarizing test pulse. The steady-state inactivation-activation curves of Ba2+ current were not significantly shifted along the voltage axis.6. The present data suggest the existence of two distinct dihydropyridine binding sites which can be bound preferentially by agonist or antagonist derivatives.

Fenoverine inhibition of calcium channel currents in single smooth muscle cells from rat portal vein and myometrium.

1. The effects of fenoverine, an antispasmodic drug, have been studied on the Ca2+ channel currents of isolated cells from rat portal vein and pregnant myometrium by the patch-clamp technique (whole-cell configuration). 2. Fenoverine inhibited both fast and slow Ca2+ channel currents in a concentration-dependent manner. Half-inhibition of fast Ca2+ channel current (holding potential of -70 mV) and slow Ca2+ channel current (holding potential of -40 mV) in portal vein smooth muscle were obtained at concentrations of 7.5 and 1.9 microM, respectively. In myometrium, the fenoverine concentration which blocked 50% of the slow Ca2+ channel current (holding potential of -70 mV) was 2.3 microM. 3. Administration of fenoverine at rest reduced both Ca2+ channel currents. Currents activated repetitively, at a rate between 0.05 and 0.1 Hz, were inhibited equally which indicates an absence of use-dependent inhibition. 4. When cells held at depolarized membrane potentials at which fast or slow Ca2+ channel currents were strongly inactivated, the inhibitory effects of fenoverine were enhanced on both Ca2+ channel currents which indicates that the fenoverine-induced inhibition was voltage-dependent. The fenoverine concentrations which blocked the inactivated Ca2+ channels were 5-7 times lower than those which blocked the resting Ca2+ channels. 5. Our results show that fenoverine depresses inward currents through fast and slow Ca2+ channels. This effect may be explained by the preferential binding of fenoverine to resting Ca2+ channels. In addition, fenoverine has a higher affinity for inactivated Ca2+ channels than for resting channels.

Identification and properties of voltage-sensitive sodium channels in smooth muscle cells from pregnant rat myometrium.

Saturable high and low affinity binding sites for [3H]saxitoxin were identified in myometrial membranes of pregnant rats, with dissociation constants of 0.53 and 27 nM, respectively. The maximal binding capacity of the low affinity binding sites was about 10 times higher than that of the high affinity binding sites. The dissociation constants obtained from association and dissociation kinetics of [3H]saxitoxin were similar to those obtained from equilibrium binding. Saxitoxin and tetrodotoxin specifically displaced [3H]saxitoxin binding at both types of sites. Isradipine (1-10 microM) and amiloride (50-100 microM) were without effect on the binding of [3H]saxitoxin. At high concentrations (10-100 microM), veratridine induced a partial inhibition of [3H]saxitoxin binding. In dispersed myometrial cells, [3H]saxitoxin binding revealed the presence of both high and low affinity binding sites, with KD values similar to those obtained in myometrial membranes. Sodium currents were studied in both freshly dispersed and cultured myometrial cells in the presence of veratridine (100 microM), using the whole-cell patch-clamp technique. Steady state inactivation curves indicated that sodium channels were available at negative membrane potentials (between -110 and -40 mV). Isradipine (1-10 microM) and amiloride (50-100 microM) were without effect on the sodium current. Applications of saxitoxin or tetrodotoxin inhibited the amplitude of the sodium current in a concentration-dependent manner. The concentrations of saxitoxin and tetrodotoxin producing half-maximal inhibition were 1.4 and 8.8 nM, respectively. Although the IC50 values for saxitoxin and tetrodotoxin found from electrophysiological experiments are not identical to the equilibrium dissociation constants for the high and low affinity sites found from binding experiments, these results suggested that binding of the neurotoxins to the high affinity sites may be involved in their inhibitory effects on sodium channels. Furthermore, low affinity binding sites may be associated with a non-functional subtype of sodium channels in myometrial cells.