Angiotensin II desensitization and Ca2+ and Na+ fluxes in vascular smooth muscle cells

The role of ion fluxes in angiotensin II (AII) desensitization (tachyphylaxis) was investigated by studying Na⁺ and Ca²⁺ translocation in cultured vascular smooth muscle cells from the rat aorta. The effects of AII were compared to those of [1-sarcosine]-AII (Sar¹-AII), an analogue which also induces tachyphylaxis, and [2-lysine]-AII (Lys²-AII), an analogue that does not show this property. Maximally effective concentrations of the three peptides induced a rapid and transient increase in ⁴⁵Ca²⁺ efflux, a rapid and sustained decrease in total cell Ca²⁺ and an increased Na⁺ permeability. Repeated treatments, at short intervals, with either of the three peptides abolished the effect on Ca²⁺ efflux, and this desensitization was slowly reversible. A 30-min rest period was sufficient for full recovery of the response of cells that were desensitized by Lys²AII, whereas the recovery from AII or Sar¹AII-desensitization was still not complete after 60 min. Our results suggest that the difference in the behaviour of the tachyphylactic AII and Sar¹-AII and the non-tachyphylactic Lys²-AII lays not in the production of different signals upon binding to the receptor, but in a difference in the hormone-receptor interaction itself.     

Modulation of large conductance Ca2+-activated K+ channel by Gαh (transglutaminase II) in the vascular smooth muscle cell

 Among G-proteins, G_h is unique in structural differences in the GTP-binding domain and possessing transglutaminase activity. We have studied the role of G protein in modulation of large conductance Ca²⁺-activated K⁺ (Maxi-K⁺) channel by the inside-out mode of patch clamp in smooth muscle cells from superior mesenteric artery of the rabbit. When the non-hydrolyzable GTP analogue, GTPγS, was applied, the channel activity was increased about 2.5-fold. Addition of GDPβS resulted in reversal of the GTPγS effect. When the Gα_h7 antibody was applied, the GTPγS-stimulated channel activity was significantly inhibited to control level, suggesting that Gα_h is involved in activation of the Maxi-K⁺ channel in smooth muscle cells. Received: 23 September 1996 / Received after revision: 26 November 1996 / Accepted: 3 December 1996     

Identification of the Ca2+ current activated by vasoconstrictors in vascular smooth muscle cells

The noncontractile aortic cell line A7r5 was chosen to study the effect of the vasoconstrictor peptide vasopressin on transmembrane Ca²⁺ movements, using conventional whole-cell patch recording techniques. Conditions in which previously characterised vasoconstrictor-modulated currents were suppressed revealed a tiny inward current component (−18±2 pA,n=50, at -61 mV in 110 mM CaCl₂). The vasopressin-activated inward current was absent when Ca²⁺ was absent from the extracellular solution, and the current amplitude increased with [Ca²⁺] (0.01–110 mM), with an apparent dissociation constant for Ca²⁺ of 9.7 mM. It was highly selective for Ca²⁺ over monovalent cations (permeability ratio Ca/Cs greater than 17). It was not voltage gated, except that the current/potential characteristic showed some inwards rectification. Amplitudes of the evoked inward currents had the same order of magnitude in Sr²⁺ and Ca²⁺, whereas they were much smaller in Mn²⁺, suggesting that this pathway is highly permeable to Sr²⁺ but poorly permeable to Mn²⁺. Inward currents evoked in Ca²⁺ were inhibited by other cations with the following order of potency: La³⁺>Cd²⁺>Co²⁺∼Ni²⁺∼Mn²⁺. The channel producing this current corresponds most probably to the ionic pathway originally called the receptor-operated calcium channel, which produces a long-lasting, constrictor-induced plateau of increased intracellular free calcium concentration in smooth muscle. Dedicated by Catherine Van Renterghem to her father, Jacques Van Renterghem     

Cellular stimulation via CD95 involves activation of phospho-inositide-3-kinase

 Several distinct intracellular pathways have been recently shown to be activated during CD95/Fas/APO-1-mediated apoptosis. Here, we demonstrate that CD95 ligation induces a rapid and transient tyrosine phosphorylation and activation of phosphoinositide-3-kinase (PI-3-K) in Jurkat T lymphocytes or CD95-sensitive glioma cells. Experiments using p56lck-deficient or p56lck-reconstituted Jurkat clones and the tyrosine kinase inhibitor herbimycin A revealed that tyrosine phosphorylation and activation of PI-3-K by CD95 depends on expression of Src-like tyrosine kinases, in particular p56lck. PI-3-K stimulation seems to be critical for CD95 receptor signalling since, first, inhibition of PI-3-K prevents CD95-mediated apoptosis and, second, CD95 receptor ligation fails to induce tyrosine phosphorylation or activation of PI-3-K in CD95-resistant glioma cells. Thus, PI-3-K activation may be an early signalling event during CD95-induced apoptosis, and failure to stimulate PI-3-K may predict tumor cell resistance to CD95-triggered apoptosis. Received: 14 May 1997 / Received after revision: 28 October 1997 / Accepted: 29 October 1997     

Modulation of Ca2+-activated K+ channels of human erythrocytes by endogenous cAMP-dependent protein kinase

 Single Ca²⁺-activated K⁺ channels of human erythrocytes were studied with the patch-clamp technique, to identify the mechanisms of their modulation by phosphorylation. In the cell-attached configuration, the openings of these channels were infrequent, as expected by the low cell Ca²⁺ content. After patch excision, the activity increased to levels determined by the Ca²⁺ concentration (0.5–10 μM) in the bath solution, then the channel activity ran down within a few minutes, to reach values of open probability lower than 0.10. The perfusion of the patch with MgATP increased the channel activity, with delayed and variable effects. Furthermore, the application of a mixture of cAMP (1 mM), MgATP (1 mM) and theophylline (1 mM) to the cytoplasmic side of excised patches led to dramatic enhancement of channel activity, which appeared within 20–120 s and decayed in tens of seconds after wash-out. The activation of the channel by the mixture was reversibly blocked by PKI_5–24, a peptide inhibitor specific to cAMP-dependent protein kinase (PKA). The level of activation promoted by cAMP and ATP was dependent on the Ca²⁺ concentration in the bathing solution. These results provide direct evidence that an endogenous PKA modulates the calcium sensitivity of Ca²⁺-activated K⁺ channels of human erythrocytes. Received: 19 February 1998 / Received after revision: 14 April 1998 / Accepted: 20 April 1998     

Physiological role of inward rectifier K+ channels in vascular smooth muscle cells

K⁺ channels play indispensable roles in establishing the membrane potential and in regulating the contractile tone of arterial smooth muscle cells. There are four types of K⁺ channels in arterial smooth muscle: voltage-dependent K⁺ (K_V), Ca²⁺-dependent K⁺ (BK_Ca), ATP-dependent K⁺ (K_ATP), and inward rectifier K⁺ (Kir2) channels. Comparatively few physiological studies have focused on Kir2 channels because they are present only in certain small-diameter cerebral and submucosal arterioles and in coronary arterial smooth muscle. Here, we review the characteristics and regulation of Kir2 channels in vascular arterial smooth muscle. Current knowledge of the predominant Kir2 channel subtype is Kir2.1, not Kir2.2 and 2.3. Electrophysiological measurements to determine the current–voltage relationship in arterial smooth muscle revealed inward rectification with a single-channel conductance of 21 pS. Kir2 channels were found to influence the resting tone of cerebral and coronary arteries based on the fact that barium (Ba²⁺) induces the constriction of these arteries at resting tone. Kir2 channels are also highly responsive to vasoconstrictors and vasodilators. For example, the vasoconstrictors endothelin-1 and angiotensin II inhibit Kir2 channel function by activating protein kinase C (PKC), and the vasodilator adenosine stimulates Kir2 channel function by increasing the level of cAMP, which subsequently activates protein kinase A (PKA). Certain pathological conditions such as left ventricular hypertrophy are associated with a decrease in Kir2 channel expression. Although our understanding of the physiological role and regulation of Kir2 channels is incomplete, it is believed that Kir2 channels contribute to the control of vascular tone in small-diameter vessels via various intracellular signalling pathways that regulate cell membrane potential.     

Neomycin inhibits K+-induced force and Ca2+ channel current in rat arterial smooth muscle

 The techniques of small vessel isometric myography and patch clamp were used to investigate the action of neomycin on K⁺-induced isometric force and voltage-gated Ca²⁺ channel currents in rat arterial smooth muscle. Neomycin and the dihydropyridine (DHP) Ca²⁺ channel antagonist (–)202–791 concentration-dependently and reversibly inhibited 40 mM K⁺-induced isometric force in rings of rat mesenteric and basilar arteries (IC₅₀ values of 70 μM and 1.2 nM, respectively, n = 10 and 4). Elevation of [Ca²⁺]_o by a factor of 2 significantly reduced the IC₅₀ values for inhibition of K⁺-induced force for both neomycin and (–)202–791 (192 μM and 3.7 nM, respectively, n = 6 and 4), but did not affect the Hill coefficient of the concentration/effect relationships. In patch-clamp experiments using freshly isolated basilar arterial myocytes, the voltage-gated inward current carried by Ba²⁺ was reversibly and concentration-dependently inhibited by neomycin (IC₅₀ 32 μM, n = 3). The concentration/effect curve for inhibition of the inward Ba²⁺ current by neomycin was significantly shifted to the right when [Ba²⁺]_o was raised from 1.8 mM to 10 mM (IC₅₀ 144 μM, n = 8). Our findings suggest that neomycin relaxes high-K⁺-induced force in rat isolated mesenteric and basilar arteries largely by inhibition of voltage-dependent and DHP-sensitive Ca²⁺ channels. Received: 1 August 1996 / Received after revision and accepted: 11 September 1996     

Characterisation of a mutant of barnacle troponin C lacking Ca2+-binding sites at positions II and IV

 This study investigates a mutant barnacle troponin C (TnC) protein (BTnC2–4-) in which the Ca²⁺-binding sites (sites II and IV) have been rendered non-functional. Eliminating Ca²⁺ binding at both Ca²⁺-binding sites of barnacle TnC did not prevent the incorporation of BTnC2–4- into TnC-depleted myofibrillar bundles, although, as expected, the mutant was not able to effect muscle regulation. We conclude that the Mg²⁺ involved in stabilising the interaction between TnC and TnI in the barnacle must bind at a separate location to the Ca²⁺-binding sites. Competition experiments between BTnC2–4- and wild-type barnacle TnC (BTnCWT) or the native isoform BTnC₂ indicate that BTnC2–4- has an approximately fourfold higher affinity for barnacle TnI than BTnCWT but a lower affinity for TnI compared to BTnC₂. These results indicate that disabling sites II and IV changes the affinity of BTnC2–4- for TnC-denuded barnacle myofibrils, altering the stability of the bond formed between TnC and the thin filament. Received: 30 September 1998 / Received after revision: 12 February 1999 / Accepted: 15 February 1999     

Effects of temperature on human L-type cardiac Ca2+ channels expressed in Xenopus oocytes

 Temperature normally affects peak L-type Ca²⁺ channel (CaCh) current with a temperature coefficient (Q ₁₀) of between 1.8 and 3.5; in cardiomyocytes attenuating protein kinase A activity increases Q ₁₀ whilst activating it lowers Q ₁₀. We examine temperature effects using cloned human cardiac CaChs expressed in Xenopus oocytes. Peak inward currents (I _Ba) through expressed CaChs (i.e. α_1Cα₂/δ_aβ_1b) exhibited a Q ₁₀ of 5.8±0.4 when examined between 15 and 25°C. The nifedipine-sensitive I _Ba exhibited a higher Q ₁₀ of 8.7±0.5, whilst the nifedipine-insensitive I _Ba exhibited Q ₁₀ of 3.7±0.3. Current/voltage (I/V) relationships shifted to negative potentials on warming. Using instead a different CaCh β subunit isoform, β_2c, gave rise to an I _Ba similar to those expressed using β_1b. We utilized a carboxyl deletion mutant, α_1C-Δ1633, to determine the temperature sensitivity of the pore moiety in the absence of auxiliary subunits; I _Ba through this channel exhibited a Q ₁₀ of 9.3±0.3. However, the Q ₁₀ for macroscopic conductance was reduced compared to that of heteromeric channels; decreasing from 5.0 (i.e. α_1Cα₂/δ_aβ_1b) and 3.9 (i.e. α_1Cα₂/δ_aβ_2c) to 2.4 (α_1C-Δ1633). These observations differ markedly from those made in studies of cardiomyocytes, and suggest that enhanced sensitivity may depend on the membrane environment, channel assembly or other regulatory factors. Received: 16 December 1997 / Accepted: 23 February 1998     

Complete reversal of run-down in rabbit cardiac Ca2+ channels by patch-cramming in Xenopus oocytes; partial reversal by protein kinase A

 The rabbit cardiac Ca²⁺ channel (α_1C) expressed in Xenopus oocytes exhibited a complete run-down of ionic currents when cell-attached patches were excised. The α_1C channel was expressed alone or was coexpressed with the accessory β_2a or β_1b subunit. The catalytic subunit of protein kinase A (PKAc) and MgATP were capable of delaying the run-down of single-channel currents. In 33% of the α_1C patches, and 26% of the α_1C+β_2a patches, inclusion of PKAc in the bath solution delayed the run-down for a maximum of 20 min. In experiments where PKAc in the bath was not sufficient to delay the run-down of channel activity, insertion of the patch back into the oocyte (patch-cramming) could restore channel activity. Gating currents were also measured in the α_1C+β_1b channel and were not subject to any run-down, even after the complete run-down of ionic currents. The results presented here reveal that PKAc is capable of delaying the run-down of currents in a subset of patches. The patch-cramming results suggest that a cytoplasmic factor, in addition to phosphorylation of the channel (by PKAc), may be involved in the maintenance of channel activity. Received: 29 October 1998 / Accepted: 4 January 1999     

Na+/Ca2+ exchange during Ca2+ repletion is not a prerequisite for the Ca2+ paradox in isolated rat hearts

 Ca²⁺ paradox damage has been suggested to be determined by Na⁺ entry during Ca²⁺ depletion and exchange of Na⁺ for Ca²⁺ during Ca²⁺ repletion. Since previously a Ca²⁺ paradox without prior increase of total intracellular [Na⁺] ([Na⁺]_i) has been observed, we investigated whether local accumulation of Na⁺ close to the inner side of the sarcolemma during Ca²⁺ depletion plays a role in the Ca²⁺ paradox by replacing all extracellular Na⁺ by Li⁺ 5 min before and during 10 min Ca²⁺-free perfusion (37°C) in isolated rat hearts (group I). Subsequently, hearts were perfused with a standard, Na⁺- and Ca²⁺-containing solution. Verapamil was used to prevent contracture due to the absence of Na⁺/Ca²⁺ exchange during Na⁺-free perfusion in the presence of Ca²⁺. In group II, the Ca²⁺-free period was omitted, and in group III normal extracellular [Na⁺] was used throughout. ²³Na-NMR was used to monitor intra- and extracellular Na⁺ signals. Total creatine kinase release was 2,977±413, 36±24 and 3170±297 IU/g dry weight in groups I, II and III respectively, indicating a full Ca²⁺ paradox in groups I and III. [Na⁺]_i decreased from 11.3±0.6 mM during control perfusion to 1.2±0.4 mM after 10 min Ca²⁺ depletion in group I, whereas in group III [Na⁺]_i was 10.9±2.2 mM during control perfusion and did not change significantly after 10 min Ca²⁺-free perfusion. It is concluded that accumulation of Na⁺ close to the inner side of the sarcolemma during Ca²⁺ depletion is not a prerequisite for the Ca²⁺ paradox. Received: 2 February 1998 / Received after revision: 31 March 1998 / Accepted: 9 April 1998     

Sodium nitroprusside and cGMP decrease Ca2+ channel availability in basilar artery smooth muscle cells

 We studied the effect of the nitric oxide (NO) donor, sodium nitroprusside (SNP), on the macroscopic and single-channel currents due to the 22-pS Ca²⁺ channel in smooth muscle cells from guinea pig basilar artery. In nystatin-perforated whole-cell recordings, 50 nM SNP decreased the macroscopic current to 63±12% of control values, without changing the voltage dependence of the current. In cell-attached patches with BAY-K8644 in the pipette, SNP caused a comparable decrease in single-channel availability (n ·P _o) that was dose dependent over the range of 10 nM to 10 μM SNP. SNP had no effect on single-channel properties, including slope conductance, voltage dependence of activation, the number of open states, the time constants of the open states, and the proportion of time spent in each open state. The effect of SNP (50 nM) on single Ca²⁺ channel openings was reproduced by 8-Br-cGMP (100 μM), which also reduced channel availability without altering channel properties. The protein kinase inhibitor H-8 (1.5 μM), which exhibits relative specificity for cGMP-dependent protein kinase, completely inhibited the decrease in single-channel availability expected with SNP. The dose-dependent decrease in Ca²⁺ channel availability caused by SNP was not altered by prior application of 8-Br-cAMP or forskolin, both of which cause an increase in Ca²⁺ channel availability in these cells. Our findings suggest that NO decreases openings of Ca²⁺ channels in basilar artery smooth muscle cells without altering channel properties, and that it does so by a mechanism likely to involve cGMP-dependent protein kinase. Received: 2 July 1996 / Received after revision: 30 September 1996 / Accepted: 2 October 1996     

Ca2+-dependent capacitance increases in rat basophilic leukemia cells following activation of store-operated Ca2+ entry and dialysis with high-Ca2+-containing intracellular solution

 Ca²⁺-dependent vesicular fusion was studied in single whole-cell patch-clamped rat basophilic leukemia (RBL) cells using the capacitance technique. Dialysis of the cells with 10 μM free Ca²⁺ and 300 μM guanosine 5′-O-(3-thiotriphosphate) (GTP[γ-S]) resulted in prominent capacitance increases. However, dialysis with either Ca²⁺ (225 nM to 10 μM) or GTP[γ-S] alone failed to induce a capacitance change. Under conditions of weak Ca²⁺ buffering (0.1 mM EGTA), activation of Ca²⁺-release-activated Ca²⁺ (CRAC) channels by dialysis with inositol 1,4,5-trisphosphate (InsP ₃) failed to induce a capacitance increase even in the presence of GTP[γ-S]. However, when Ca²⁺ATPases were inhibited by thapsigargin, InsP ₃ and GTP[γ-S] led to a pronounced elevation in membrane capacitance. This increase was dependent on a rise in intracellular Ca²⁺ because it was abolished when cells were dialysed with a high level of EGTA (10 mM) in the recording pipette. The increase was also dependent on Ca²⁺ influx because it was effectively suppressed when external Ca²⁺ was removed. Our results demonstrate that I _CRAC represents an important source of Ca²⁺ for triggering a secretory response. Received: 1 May 1998 / Received after revision: 15 June 1998 / Accepted: 2 July 1998     

Effects of Angiotensin II on sustained outward currents in rat ventricular myocytes

We investigated the effects of angiotensin II (Ang II) on the sustained outward current (I _sus) and action potential of rat ventricular myocytes using the whole-cell patch-clamp technique. Ang II at 30 nM~3 µM inhibited I _sus with an IC₅₀ of 240 nM, a Hill coefficient of 1.0 and maximum inhibition of 19.4%. Ang II-mediated inhibition of I _sus was voltage independent, was due to a decrease in the K⁺ current and was abolished by the Ang II type-I (AT₁) receptor blocker, valsartan. The protein kinase C (PKC) inhibitors PKC19–36 or calphostin C, abolished Ang II-mediated inhibition of I _sus. In contrast, pretreatment with the protein kinase A (PKA) inhibitor PKA6–22 (100 µM) significantly enhanced the suppression of I _sus by 1 µM Ang II: (33.7±5.1% vs. control 17.1±2.3%). These results indicate that Ang II inhibits I _sus via the AT₁ receptor and activation of PKC. Ang II significantly prolonged action potential duration (APD) when the control APD was lengthened by a Ca²⁺ channel activator, BAY K8644. In myocytes with a relatively long APD, Ang II may prolong APD by inhibiting I _sus.     

Inhibition of the L-type calcium channel by the five muscarinic receptors (m1–m5) expressed in NIH 3T3 cells

 Modulation of L-type calcium channels by the five cloned muscarinic receptors was studied by expression of the receptors in NIH 3T3 cells. Application of acetylcholine (ACh) to cells transfected with m1–m5 resulted in a reduction in the L-type calcium current amplitude. Elevations in intracellular cAMP concentrations induced by 8-bromo-cAMP or forskolin resulted in no discernible change in the L-type calcium current. In addition, treatment with Rp-adenosine 3′,5′-cyclic monophosphothioate triethylamine (Rp-cAMPS), a protein kinase A (PKA) inhibitor, had no effect on the L-type currents. Conversely, application of phorbol dibutyrate, an activator of protein kinase C (PKC) or 8-bromo-cGMP, an activator of cGMP-dependent protein kinase (PKG), reduced the calcium currents. Incubation of the cells with KT5823, an inhibitor of PKG, resulted in a reduction of the response to 8-bromo-cGMP. The ACh-induced depression of L-type calcium current amplitude was sensitive to pertussis toxin (PTX) in cells transfected with the m2 or m4 receptor subtype. The m2-muscarinic-receptor-induced inhibition of the L-type calcium current was attenuated by preincubation of the cells with 8-bromo-cAMP and was unaffected by KT5823 or by calphostin C. The m1-muscarinic-receptor-induced inhibition of the L-type calcium conductance was insensitive to PTX treatment. However, the m1-induced response was blocked by preincubation of the cells with calphostin C. The present data indicate that the m2 (and possibly also the m4) muscarinic receptors inhibit the L-type calcium conductance by a reduction in cAMP concentration and that the m1 (and possibly also the m3 and m5) muscarinic receptors inhibit the L-type calcium channel via activation of PKC. Received: 2 September 1996 / Received after revision and accepted: 15 October 1996     

Ca2+ and K+ current in cultured vascular smooth muscle cells from rat aorta

Ca²⁺ (I_Ca) and K⁺ (I_K) currents were recorded in single cultured cells from rat aorta using the whole cell clamp technique with patch electrodes. I_Ca was detected at–30 mV, and at 20 mV it reached a peak in about 10 ms and decayed with a t_1/2=50 ms. The mean maximum slope conductance (G_Ca) was 30 S/cm². I_K was detected at–10 mV and at 20 mV reached its maximum with a t_1/2=12 ms. For I_K, G_K=200 S/cm². These channels can be activated during action potentials and play a role in the excitation and contraction of vascular smooth muscle cells.Doctoral training program UAM-1     

Effects of the protein kinase A inhibitor H-89 on Ca2+ regulation in isolated ferret ventricular myocytes

 We investigated the effects of a protein kinase A (PKA) inhibitor, H-89 {N-[2-(p-bromocinnamylamino)ethyl]-5-iso-quinolinesulphonamide}, on Ca²⁺ regulation in Fura-2-loaded ferret myocytes. H-89 (10 μmol/l) decreased the amplitude of the Fura-2 transient to 28.2±4.3% (P<0.001) of control and prolonged its duration, characterized by a decrease in the rate of decline of Ca²⁺ to diastolic levels: t _1/2 increased from 311±35 ms to 547±43 ms (P<0.001, n=7). Reduced Ca²⁺ uptake by the sarcoplasmic reticulum (SR) in the presence of H-89 was also indicated by a decrease in the SR Ca²⁺ content, as assessed with caffeine. The apparent slowing of the SR Ca²⁺-ATPase was not caused by changes in phosphorylation of phospholamban (PLB). However, Ca²⁺ uptake in microsomal vesicles prepared from canine hearts and fast-twitch rat skeletal muscle (which lacks PLB) was decreased by 34.1 and 46.8% (n=3), respectively, suggesting that H-89 has a direct inhibitory effect on the SR Ca²⁺-ATPase. In electrophysiological experiments, 5.0 μmol/l H-89 decreased the L-type Ca²⁺ current (I _Ca) by 39.5% (n=6) and slowed the upstroke of the action potential and, in some cases, caused loss of excitability without changes in the resting membrane potential. In summary, data show that [Ca²⁺ ]_i regulation, and hence contraction, is sustained by PKA-mediated phosphorylation, even in the absence of β-agonists. However, the use of H-89 as a tool to study the role of this signalling pathway is limited by the non-specific effects of H-89 on the SR Ca²⁺-ATPase. Received: 4 September 1998 / Received after revision: 19 October 1998 / Accepted: 20 October 1998     

Simultaneous measurements of electrical activity, intracellular [Ca2+] and force in intact smooth muscle

 Although both electrical activity and changes in intracellular [Ca²⁺] are known to be important determinants of smooth muscle force, little is known about the relationship between the three in intact muscle. This is due to a lack of simultaneous measurements of these parameters. In this paper we describe how we have combined the sucrose gap technique with microspectrophotometry and force recording in rat ureteric smooth muscle. We have investigated the timecourses of the changes in these parameters following physiological stimulation i.e. the action potential. The results of this paper show that it is possible to simultaneously measure electrical activity, [Ca] and force in intact smooth muscle and that (i) about a third of the change in Ca²⁺ occurs on the upstroke of the action potential and the remainder during the plateau. (ii) 50% of the decline in [Ca²⁺] occurs after the repolarization of the action potential and (iii) the kinetics of force development and relaxation are significantly slower than those of Ca²⁺, suggesting that [Ca²⁺] is not rate-limiting. These are the first such simultaneous measurements in any smooth muscle. Received: 31 July 1997 / Received after revision: 9 September 1997 / Accepted: 12 September 1997     

Ca2+ entry through cardiac L-type Ca2+ channels modulates β-adrenergic stimulation in mouse ventricular myocytes

 β-adrenergic receptor (β-AR) stimulation increases cardiac L-type Ca²⁺ channel (CaCh) currents via cAMP-dependent phosphorylation. We report here that the affinity and maximum response of CaCh to isoproterenol (Iso), in mouse ventricular myocytes were significantly higher when Ba²⁺ was used as the charge carrier (I _Ba) instead of Ca²⁺ (I _Ca). The EC₅₀ and maximum increase of peak currents were 43.7 ± 7.9 nM and 1.8 ± 0.1-fold for I _Ca and 23.3 ± 4.7 nM and 2.4 ± 0.1-fold for I _Ba. When cells were dialyzed with the faster Ca²⁺ chelator, BAPTA, both sensitivity and maximum response of I _Ca to Iso were significantly augmented compared to cells with EGTA (EC₅₀ of 23.1 ± 5.2 nM and maximal increase of 2.2 ± 0.1-fold). Response of I _Ca to forskolin was also significantly increased when cells were dialyzed with BAPTA or when currents were measured in Ba²⁺. In contrast, depletion of the sarcoplasmic reticulum (SR) Ca²⁺ stores by ryanodine did not alter sensitivity of I _Ca to Iso or forskolin. These results suggest that the Ca²⁺ entering through CaCh regulates cAMP-dependent phosphorylation, and such negative feedback may play a significant role in cellular Ca²⁺ homeostasis and contraction in cardiac cells during β-AR stimulation. Received: 10 December 1997 / Received after revision: 19 January 1998 / Accepted: 21 January 1998     

Effect of adrenergic agonists on Ca2+-channel currents in single vascular smooth muscle cells

Ca²⁺-channel currents have been measured in enzymatically dispersed single smooth muscle cells of the rabbit ear artery using the whole-cell patch clamp technique. Inward currents were elicited by depolarizing test pulses from a holding potential of–50 mV. These currents were activated from–30 mV onward and reached full activation around 0 mV. -Adrenergic agonists did not affect the background current measured at the holding potential, but markedly reduced the peak amplitude of the voltageactivated Ca²⁺-channel currents. This -adrenergic inhibition also occurred in cells which were internally perfused with solutions containing either 10 M cAMP, 10M cGMP or 0.1 mM GTP, but became irreversible when the pipette solution contained a non-hydrolyzable GTP-analog. The action of -agonists on the voltage-activated Ca²⁺-channel currents was variable, and ranged from no effect at all to a 50% reduction of the current. It is concluded that -agonists do not open receptor-operated Ca²⁺-channels in these smooth muscle cells. The inhibition of the voltageactivated Ca²⁺-currents does not seem to be mediated through changes in cyclic nucleotide levels, but might be mediated through G-proteins. Its physiological relevance remains however unclear. The action of -agonists is consistent with their relaxing effect, but the reason for the nonuniform response has not been elucidated.