Stimulatory actions of di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS), voltage-sensitive dye, on the BKCa channel in pituitary tumor (GH3) cells

Di-8-ANEPPS (4-{2-[6-(dibutylamino)-2-naphthalenyl]-ethenyl}-1-(3-sulfopropyl)pyridinium inner salt) has been used as a fast-response voltage-sensitive styrylpyridinium probe. However, little is known regarding the mechanism of di-8-ANEPPS actions on ion currents. In this study, the effects of this dye on ion currents were investigated in pituitary GH₃ cells. In whole-cell configuration, di-8-ANEPPS (10 μM) reversibly increased the amplitude of Ca²⁺-activated K⁺ current. In inside-out configuration, di-8-ANEPPS (10 μM) applied to the intracellular surface of the membrane caused no change in single-channel conductance; however, it did enhance the activity of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels with an EC₅₀ value of 7.5 μM. This compound caused a left shift in the activation curve of BK_Ca channels with no change in the gating charge of these channels. A decrease in mean closed time of the channels was seen in the presence of this dye. In the cell-attached mode, di-8-ANEPPS applied on the extracellular side of the membrane also activated BK_Ca channels. However, neither voltage-gated K⁺ nor ether-à-go-go-related gene (erg)-mediated K⁺ currents in GH₃ cells were affected by di-8-APPNES. Under current-clamp configuration, di-8-ANEPPS (10 μM) decreased the firing of action potentials in GH₃ cells. In pancreatic βTC-6 cells, di-8-APPNES (10 μM) also increased BK_Ca-channel activity. Taken together, this study suggests that during the exposure to di-8-ANEPPS, the stimulatory effects on BK_Ca channels could be one of potential mechanisms through which it may affect cell excitability.     

The effect of the outermost basic residues in the S4 segments of the CaV3.1 T-type calcium channel on channel gating

The contribution of voltage-sensing S4 segments in domains I to IV of the T-type Ca_V3.1 calcium channel to channel gating was investigated by the replacement of the uppermost charged arginine residues by neutral cysteines. In each construct, either a single (R180C, R834C, R1379C or R1717C) or a double (two adjacent domains) mutation was introduced. We found that the neutralisation of the uppermost arginines in the IS4, IIS4 and IIIS4 segments shifted the voltage dependence of channel activation in a hyperpolarising direction, with the most prominent effect in the IS4 mutant. In contrast, the voltage dependence of channel inactivation was shifted towards more negative membrane potentials in all four single mutant channels, and these effects were more pronounced than the effects on channel activation. Recovery from inactivation was affected by the IS4 and IIIS4 mutations. In double mutants, the effects on channel inactivation and recovery from inactivation, but not on channel activation, were additive. Exposure of mutant channels to the reducing agent dithiothreitol did not alter channel properties. In summary, our data indicate that the S4 segments in all four domains of the Ca_V3.1 calcium channels contribute to voltage sensing during channel inactivation, while only the S4 segments in domains I, II and III play such role in channel activation. Furthermore, the removal of the outermost basic amino acids from the IVS4 and IIIS4 and, to a lesser extent, from IS4 segments stabilised the open state of the channel, whereas neutralization from that of IIS4 destabilised it. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Martina Kurejová and L’ubica Lacinová contributed equally to this work. An erratum to this article can be found at     

Store-operated Ca2+ entry in platelets occurs independently of transient receptor potential (TRP) C1

Changes in [Ca²⁺]_i are a central step in platelet activation. In nonexcitable cells, receptor-mediated depletion of intracellular Ca²⁺ stores triggers Ca²⁺ entry through store-operated calcium (SOC) channels. Stromal interaction molecule 1 (STIM1) has been identified as an endoplasmic reticulum (ER)-resident Ca²⁺ sensor that regulates store-operated calcium entry (SOCE), but the identity of the SOC channel in platelets has been controversially debated. Some investigators proposed transient receptor potential (TRP) C1 to fulfil this function based on the observation that antibodies against the channel impaired SOCE in platelets. However, others could not detect TRPC1 in the plasma membrane of platelets and raised doubts about the specificity of the inhibiting anti-TRPC1 antibodies. To address the role of TRPC1 in SOCE in platelets, we analyzed mice lacking TRPC1. Platelets from these mice display fully intact SOCE and also otherwise unaltered calcium homeostasis compared to wild-type. Furthermore, platelet function in vitro and in vivo is not altered in the absence of TRPC1. Finally, studies on human platelets revealed that the presumably inhibitory anti-TRPC1 antibodies have no specific effect on SOCE and fail to bind to the protein. Together, these results provide evidence that SOCE in platelets is mediated by channels other than TRPC1. David Varga-Szabo and Kalwant S. Authi contributed equally to this article.     

Caffeine and histamine-induced oscillations of K(Ca) current in single smooth muscle cells of rabbit cerebral artery

In the present experiment, we characterized the intracellular Ca²⁺ oscillations induced by caffeine (1 mM) or histamine (1–3 M) in voltage-clamped single smooth muscle cells of rabbit cerebral (basilar) artery. Superfusion of caffeine or histamine induced periodic oscillations of large whole-cell K⁺ current with fairly uniform amplitudes and intervals. The oscillatory K⁺ current was abolished by inclusion of ethylenebis(oxonitrilo)tetraacetate (EGTA, 5 mM) in the pipette solution. Caffeine- and histamine-induced periodic activation of the large-conductance Ca²⁺-activated K⁺ [K_(Ca)] channel was recorded in the cell-attached patch mode. These results suggest that the oscillations of K⁺ current are carried by the K_(Ca) channel and reflect the oscillations of intracellular Ca²⁺ concentration ([Ca²⁺]_i). Ryanodine (1–10 M) abolished both caffeine- and histamine-induced oscillations. Caffeine- induced oscillations were abolished by the sarcoplasmic reticulum Ca²⁺-adenosine 5-triphosphatase (Ca²⁺-ATPase) inhibitor, cyclopiazonic acid (10 M), and a high concentration of caffeine (10 mM). Inclusion of heparin (3 mg/ml) in the pipette solution blocked histamine-induced oscillations, but did not block caffeine-induced oscillations. By the removal of extracellular Ca²⁺, but not by the addition of verapamil and Cd²⁺, the caffeine-induced oscillations were abolished. Increasing Ca²⁺ influx rate increased the frequencies of caffeine-induced oscillations. Spontaneous oscillations were also observed in cells that were not superfused with agonists, and had similar characteristics to the caffeine-induced oscillations. From the above results, it is concluded, that in smooth muscle cells of the rabbit cerebral (basilar) artery, ryanodine-sensitive Ca²⁺-induced Ca²⁺ release pools play key roles in the generation of caffeine- and histamine-induced intracellular Ca²⁺ oscillations.     

Functional characterization of the Ca2+-gated Ca2+ release channel of vascular smooth muscle sarcoplasmic reticulum

The Ca²⁺-gated Ca²⁺ release channel of aortic sarcoplasmic reticulum (SR) was partially purified and reconstituted into planar lipid bilayers. Canine and porcine aorta microsomal protein fractions were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate (CHAPS) in the presence and absence of ³[H]-ryanodine and centrifuged through linear sucrose gradients. A single ³[H]-ryanodine receptor peak with an apparent sedimentation coefficient of 30 s was obtained. Upon reconstitution into planar lipid bilayers, the unlabelled 30 s protein fraction induced the formation of a Ca²⁺- and monovalent-ion-conducting channel (110 pS in 100 mM Ca²⁺, 360 pS in 250 mM K⁺). The channel was activated by micromolar Ca²⁺, modulated by millimolar adenosine triphosphate, Mg²⁺ and the Ca²⁺-releasing drug caffeine, and inhibited by micromolar ruthenium red. Micro- to millimolar concentrations of the plant alkaloid ryanodine induced a permanently closed state of the channel. Our results suggest that smooth muscle SR contains a Ca²⁺-gated Ca²⁺ release pathway, with properties similar to those observed for the skeletal and cardiac ryanodine receptor/Ca²⁺ release channel complexes.     

G-protein-mediated regulation of a Ca2+-dependent K+ channel in cultured vascular endothelial cells

The purpose of the present study was to determine the mechanism by which bradykinin activates the small conductance, inwardly rectifying, Ca²⁺-activated K⁺ channel (K_Ca) found in cultured bovine aortic endothelial cells. Channel activity was studied using the patch-clamp technique in whole-cell, cell-attached, inside-out and outside-out configurations. Channel conductance at potentials positive to 0 mV was 10±2 pS and at potentials negative to 0 mV 30±3 pS (n=7) when examined in symmetrical K⁺ (150 mmol/l) solutions. The channel open probability (P _o) was only weakly voltage dependent changing approximately 0.2 units over 160 mV. In contrast, raising the intracellular Ca²⁺ concentration from 100 nmol/l to 10 mol/l at –60 mV produced a graded increase in channel P _o from 0.15 to 0.96; the concentration required for half-maximum response (apparent K_0.5) was 719 nmol/l. At a constant Ca²⁺ concentration, application of guanosine triphosphate (GTP) to the cytoplasmic surface of the patch increased channel P _o. This effect was dependent upon the simultaneous presence of both GTP and Mg²⁺, and was reversed by the subsequent application of the guanosine diphosphate (GDP) analogue, guanosine-5-O-(2-thiodiphosphate) (GDPS). The hydrolysis-resistant GTP analogue, guanosine-5-O-(3-thiotriphosphate) (GTPS), induced a long-lasting increase in channel P _o. In the presence of Mg²⁺-GTP, the apparent K_0.5 for Ca²⁺ decreased from a control value of 722 nmol/l to 231 nmol/l. Addition of bradykinin to outside-out patches previously exposed to intracellular Mg²⁺-GTP further enhanced K_Ca activity, shifting the apparent K_0.5 for Ca²⁺ from 228 nmol/l to 107 nmol/l. This activation by bradykinin was not observed in patches following prior exposure to GDPS. These results suggest that bradykinin can activate the K_Ca channel of vascular endothelial cells via a G-protein-mediated change in the sensitivity of the channel for Ca²⁺. We postulate that vasoactive agonists may use this mechanism to maintain an elevated K⁺ permeability as the intracellular Ca²⁺ concentration returns towards normal resting levels.     

Peculiarities of Ca2+-induced erythrocyte response in patients with lung cancer undergoing antitumor chemotherapy

Characteristics of changes in the membrane potential induced by increased Ca²⁺ inward current in patients with lung cancer are presented. Both the amplitude and the rate of hyperpolarization induced by opening of Ca²⁺-activated potassium channels are markedly decreased in erythrocytes from patients in comparison with healthy donors. Under conditions of moderate Ca²⁺ inward current, hyperpolarization is followed by restoration of the membrane potential. This process is more rapid in cancer patients. It is shown that the parameters of hyperpolarization, in their dynamics during antitumor therapy depend on the type of lung cancer. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 8, pp. 195–198, August, 1997     

D600 blocks the Ca2+ channel from the outer surface of smooth muscle cell membrane of the rabbit intestine and portal vein

The voltage dependent Ca²⁺ inward current in single smooth muscle cells dispersed from the longitudinal muscle layer of the rabbit ileum and rabbit portal vein was recorded using the whole-cell voltage clamp technique. D600 added to the bathing solution inhibited the Ca²⁺ current, while the intracellular perfusion of this agent did not reduce the amplitude of this current. Thus, D600 probably acts from the outer surface of the membrane. The nature of the Ca²⁺ channel in smooth muscle cells seems to differ from that in cardiac muscle cells.     

Probing a Ca2+-activated K+ channel with quaternary ammonium ions

A series of quaternary amonium (QA) ions were used to probe the gross architecture of the ion conduction pathway in a Ca²⁺-activated K⁺ channel from rat muscle membrane. The channels were inserted into planar phospholipid membranes and the single channel currents were measured in the presence of the different QA ions. Internally applied monovalent QA ions (e.g. tetramethylammonium and analogues) induced a voltage-dependent blockade with a unique effective valence of the block equal to 0.30, and blocking potency increases as the compound is made more hydrophobic. Blockade is relieved by increasing the K⁺ concentration of the internal or external side of the channel. The effective valence of block is independent of K⁺ concentration. These results suggest that, from the internal side, all monovalent QA ions interact with a site located in the channel conduction system. Divalent QA ions of the type n-alkylbis-,-trimethylammonium (bisQn) applied internally also block the channel in a voltage dependent fashion. For short chains (bisQ2-bisQ5), the effective valence decreases with chain length from 0.41 to 0.27, it remains constant for bisQ5 to bisQ6 and increases up to 0.54 for bisQ10. This dependence of block with chain length implies that 27% of the voltage drop within the channel occurs over a distance of 1 nm. Externally applied monovalent QA ions also block the channel. The site is specific for tetraethylammonium; increasing or decreasing the side chains in one methylene group decrease potency by about 400-fold. It is concluded that the Ca²⁺-activated K⁺ channel has wide mouths located at each end and that they are different in molecular nature.     

Single Ca channel currents in mouse pancreatic B-cells

Barium currents flowing through single Ca²⁺ channels were recorded from outside-out patches isolated from mouse pancreatic B-cells. Only one type of Ca²⁺ channel was observed. In 110 mM Ba²⁺, the single channel conductance was 24 pS (at negative membrane potentials) and the current amplitude at 0 mV was–0.7 pA. Channel openings were activated by depolarisations more positive than –30 mV and showed little inactivation during 200 ms pulses. Open times were increased by BAY K 8644 an decreased by micromolar Cd²⁺. Channel activity was subject to rundown in excised patches and little activity remained after 10 min. These properties resemble those of L-type Ca²⁺ channels in other tissues. It is suggested that this Ca²⁺ channel participates in the generation of the B-cell action potential and mediates the increase in Ca²⁺ influx required for insulin secretion.     

Ca2+ channel Ca2+-dependent inactivation in a mammalian central neuron involves the cytoskeleton

Ca²⁺ channel inactivation was investigated in acutely isolated hippocampal pyramidal neurons from adult rats and found to have a component dependent on intracellular Ca²⁺. Ca²⁺-dependent inactivation was identified as the additional inactivation of channel current observed when Ca²⁺ replaced Ba²⁺ as the current carrying ion, and was found to be an independent process from that of Ba²⁺ current inactivation based on three lines of evidence: (1) no correlation between Ca²⁺-dependent inactivation and Ba²⁺ current inactivation was found, (2) only Ca²⁺-dependent inactivation was reduced by intracellular application of Ca²⁺ chelators, and (3) only Ca²⁺-dependent inactivation was sensitive to compounds which alter the cytoskeleton. Drugs which stabilize (taxol and phalloidin) and destabilize (colchicine and cytochalasin B) the cytoskeleton altered the development and recovery from Ca²⁺-dependent inactivation, indicating that the neuronal cytoskeleton may mediate Ca²⁺ channel sensitivity to intracellular Ca²⁺. Ca²⁺-dependent inactivation was not associated with a particular subset of Ca²⁺ channels, suggesting that all Ca²⁺ channels in these neurons are inactivated by intracellular Ca²⁺.     

CaMKII phosphorylates a threonine residue in the C-terminal tail of Cav1.2 Ca<Superscript>2+</Superscript> channel and modulates the interaction of the channel with calmodulin

We have previously found that both CaMKII-mediated phosphorylation and calmodulin (CaM) binding to the channels are required for maintaining basal activity of the Cav1.2 Ca²⁺ channels. In this study, we investigated the hypothetical CaMKII phosphorylation site on Cav1.2 that contributes to the channel regulation. We found that CaMKII phosphorylates the Thr1603 residue (Thr1604 in rabbit) within the preIQ region in the C-terminal tail of the guinea-pig Cav1.2 channel. Mutation of Thr1603 to Asp (T1603D) slowed the run-down of the channel in inside-out patch mode and abolished the time-dependency of the CaM’s effects to reverse run-down. We also found that CaMKII-mediated phosphorylation of the proximal C-terminal fragment (CT1) increased, while dephosphorylation of CT1 decreased its binding with CaM. These findings suggest that CaMKII regulates the CaM binding to the channel, and thereby maintains basal activity of the Cav1.2 Ca²⁺ channel.     

Tetraethylammonium ion sensitivity of a 35-pS Ca2+-activated K+ channel in GH3 cells that is activated by thyrotropin-releasing hormone

Single Ca²⁺-activated K⁺ channels were studied in membrane patches from the GH₃ anterior pituitary cell line. We have previously demonstrated the coexistence of large-conductance and small-conductance (280 pS and 11 pS in symmetrical 150 mM K⁺, respectively) Ca²⁺-activated K⁺ channels in this cell line (Lang and Ritchie 1987). Here we report the existence of a third type of Ca²⁺-activated K⁺ channel that has a conductance of about 35 pS under similar conditions. In excised inside-out patches, this channel can be activated by elevations of the internal free Ca²⁺ concentration, and the open probability increases as the membrane potential is made more positive. In excised patches, the sensitivity of this 35-pS channel to internal Ca²⁺ is low; at positive membrane potentials, this channel requires Ca²⁺ concentrations greater than 10 M for activation. However, 35-pS channels have a much higher sensitivity to Ca²⁺ in the first minute after excision (activated by 1 M Ca²⁺ at –50 mV). Therefore, it is possible that the Ca²⁺ sensitivity of this channel is stabilized by intracellular factors. In cell-attached patches, this intermediate conductance channel can be activated (at negative membrane potentials) by thyrotropin-releasing hormone-induced elevations of the intracellular Ca²⁺ concentration and by Ca²⁺ influx during action potentials. The intermediate conductance channel is inhibited by high concentrations of external tetraethylammonium ions (K _d=17 mM) and is relatively resistant to inhibition by apamin.     

Correlation of Intracellular Ca2+-Activated Proteinase Activity and Cholesterol Content in White Sea Mussel (Mytilus edulis) Membranes at Different Water Saltiness

Correlation between changes in activity of intracellular Ca²⁺-activated proteinases and cholesterol content in mussels in response to changes in habitat saltiness was detected in mollusks from littoral and sublittoral White sea zones. Calpain activity in mussels decreased in low water saltiness and increased in high saltiness in parallel with decrease in cholesterol content, which attests to decreased microviscosity and modification of permeability of biomembranes. A complex pattern of interactions between metabolic routes in mussels under conditions of different habitat saltiness was detected. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 140, No. 10, pp. 457–460, October, 2005     

A small-conductance charybdotoxin-sensitive,apamin-resistant Ca2+-activated K+ channel in aortic smooth muscle cells (A7r5 line and primary culture)

A small conductance K⁺ channel was identified in smooth muscle cells of the rat aortic cell line A7r5 and also in rat aortic smooth muscle cells in primary culture, using conventional single-channel recording techniques. The single-channel conductance shows no rectification, either in the range –70 to +40 mV under asymmetrical conditions (9.1 pS), or in the range –100 to +50 mV in symmetrical 150 mM K⁺ (37 pS). Channel activity is reversibly inhibited by extracellular application of charybdotoxin, with a concentration of 8 nM producing half-maximal inhibition. It is unaffected by apamin or scyllatoxin. Channel activity depends on the presence of free Ca²⁺ on the cytosolic face of the membrane, with an activation zone between 0.1 and 1 M. This small-conductance, charybdotoxin-sensitive, Ca²⁺-regulated K⁺ channel is activated by vasoconstrictors such as vasopressin and endothelin.     

Neuropeptide Y inhibits Ca2+-activated K+ channels in vascular smooth muscle cells from the rat tail artery

The effects of neuropeptide Y (NPY) on the Ca²⁺-activated K⁺ channel in smooth muscle cells from the rat tail artery were studied by whole-cell and single-channel patch-clamp recording techniques. In the presence of nifedipine (1 M), whole-cell outward currents through Ca²⁺-activated K⁺ channels were inhibited by NPY in a dose-dependent manner from 20 to 200 nM. A maximum inhibition to about 48% of the control current could be achieved. Recordings from outside-out patches showed that the open probability of Ca²⁺-activated K⁺ channels were similarly inhibited by NPY. At 200 nM NPY, the open probability was reduced to about 36% of the control value. NPY did not affect the open times or current amplitude, but increased significantly the short (from 0.49 to 0.58 ms) and long (from 441 to 728 ms) closed times. Inhibition of Ca²⁺-activated K⁺ channels by NPY may contribute to its excitatory action on vascular smooth muscle cells.     

A novel molecular inactivation determinant of voltage-gated CaV1.2 L-type Ca2+ channel

The inactivation of voltage-gated L-type Ca(2+) channels (Ca(V)1) regulates Ca(2+) entry and controls intracellular Ca(2+) levels that are essential for cellular activity. The molecular entities implicated in L-channel (Ca(V)1.2) inactivation are not fully identified. Here we show for the first time the functional impact of one of the two highly conserved clusters of six negatively charged glutamates and aspartate (802-807; poly ED motif) at the II-III loop of the alpha 1 subunits of rabbit of Ca(v)1.2, alpha(1)1.2 and alpha(1)1.2 DeltaN60-Delta1733) on voltage-dependent inactivation. Mutation of the poly ED motif to alanine or glutamine/asparagine greatly enhanced voltage-dependent inactivation, shifting the voltage dependence to negative potentials by >50 mV and conferring a neuronal like inactivation kinetics onto Ca(V)1.2. The large shift in the midpoint of inactivation of the steady-state inactivation kinetics was observed also in Ca(2+) or Ba(2+) and was not altered by the beta2A subunit. Missing from the fast inactivating neuronal P/Q (Ca(V)2.1)-, N (Ca(V)2.2)- or R (Ca(V)2.3)-type channels and modulating Ca(V)1.2 inactivation kinetics, the poly ED motif is likely to be a specific L-type Ca(2+) channels inactivating domain. Our results fit a model in which the poly ED either by itself or as part of a larger inactivating motif acts as Ca(V)1.2 specific built-in "stopper." In this model, Ca(V)1 accomplishes a large Ca(2+) influx during depolarization, possibly by the poly ED hindering occlusion at the pore. Furthermore, the selective designed poly ED perhaps clarifies major inactivation differences between L- and non-L-type calcium channels.     

A novel method for incorporation of ion channels into a planar phospholipid bilayer which allows solution changes on a millisecond timescale

We have developed a method of rapidly changing the solutions on one side of a planar phospholipid bilayer. Bilayers can be painted on glass pipettes of tip diameter 50 m. By modifying an established method for rapid exchange of solutions bathing excised membrane patches, solution changes can be made at the bilayer within 10 ms. After incorporation of channels into the bilayer, the bilayer is moved into one of two parallel streams of solution flowing from a length of double-barrelled glass theta tubing. Activation of a solenoid system rapidly moves the theta tubing so that the bilayer is in the flow of the adjacent solution. For various reasons, the single-channel gating mechanisms of many channels are studied in planar bilayer systems. The conventional bilayer technique only allows for steady-state single-channel gating to be monitored. This novel method now allows the effects of rapid changes in modulators of channels incorporated into planar phospholipid bilayers to be measured.     

Regulation of membrane excitability by intracellular pH (pHi) changers through Ca2+-activated K+ current (BK channel) in single smooth muscle cells from rabbit basilar artery

Employing microfluorometric system and patch clamp technique in rabbit basilar arterial myocytes, regulation mechanisms of vascular excitability were investigated by applying intracellular pH (pH_i) changers such as sodium acetate (SA) and NH₄Cl. Applications of caffeine produced transient phasic contractions in a reversible manner. These caffeine-induced contractions were significantly enhanced by SA and suppressed by NH₄Cl. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was monitored in a single isolated myocyte and based the ratio of fluorescence using Fura-2 AM (R _340/380). SA (20 mM) increased and NH₄Cl (20 mM) decreased R _340/380 by 0.2 ± 0.03 and 0.1 ± 0.02, respectively, in a reversible manner. Caffeine (10 mM) transiently increased R _340/380 by 0.9 ± 0.07, and the ratio increment was significantly enhanced by SA and suppressed by NH₄Cl, implying that SA and NH₄Cl may affect [Ca²⁺]_i (p < 0.05). Accordingly, we studied the effects of SA and NH₄Cl on Ca²⁺-activated K⁺ current (IK_Ca) under patch clamp technique. Caffeine produced transient outward current at holding potential (V _h) of 0 mV, caffeine induced transient outward K⁺ current, and the spontaneous transient outward currents were significantly enhanced by SA and suppressed by NH₄Cl. In addition, IK_Ca was significantly increased by acidotic condition when pH_i was lowered by altering the NH₄Cl gradient across the cell membrane. Finally, the effects of SA and NH₄Cl on the membrane excitability and basal tension were studied: Under current clamp mode, resting membrane potential (RMP) was −28 ± 2.3 mV in a single cell level and was depolarized by 13 ± 2.4 mV with 2 mM tetraethylammonium (TEA). SA hyperpolarized and NH₄Cl depolarized RMP by 10 ± 1.9 and 16 ± 4.7 mV, respectively. SA-induced hyperpolarization and relaxation of basal tension was significantly inhibited by TEA. These results suggest that SA and NH₄Cl might regulate vascular tone by altering membrane excitability through modulation of [Ca²⁺]_i and Ca²⁺-activated K channels in rabbit basilar artery.     

Contractile activation properties of ventricular myocardium from hypothyroid,euthyroid and juvenile rats

Contractile activation properties of intact and chemically skinned ventricular myocardium preparations were studied in juvenile (3–4 weeks old), adult euthyroid and adult hypothyroid rats. The rats were made hyperthyroid by treatment with iodine-131 and propylthiouracil. The ventricular muscle of euthyroid rats contains a mixture of isozymes of myosin while the myocardium of juvenile and hypothyroid rats are relatively pure in regard to V₁ and V₃ types of myosin respectively. No significant differences were found in either the maximum Ca²⁺ activated or rigor force developed by chemically skinned preparations in either the juvenile or hypothyroid groups compared with euthyroid adults, suggesting that there is no difference between myocardia with different isozymes of myosin in the intrinsic capacity to generate force. In the hypothyroid (V₃) preparations there was a significant shift in the force/pCa relation to the left compared with the euthyroid adult (mixture of V₁ and V₃ isozymes). The force/pCa relation for the juvenile lay in between that for the hypothyroid and euthyroid adults. The greater apparent Ca²⁺ sensitivity to activation in the hypothyroid group may relate to a slower cross-bridge cycling rate or altered Ca²⁺ kinetics in ventricular myocardium with exclusively V₃ isozyme. In intact papillary muscles differences were found in the dependence efforce on extracellular [Ca²⁺] such that a higher extracellular [Ca²⁺] was required for muscles from hypothyroid animals to attain maximum twitch force than those from juveniles. The force/frequency relations also differed, with the hypothyroid group being better able to sustain force as stimulation frequency increased than the juvenile group. Also, in the hypothyroid group, the contraction following a 3-min period of quiescence was potentiated in relation to the preceding steady-state contractions, whereas in the juvenile group it was not. These results indicate that the thyroid state may influence the pattern of calcium translocation as well as the myosin isozyme type and that both of these factors may influence contractile properties. Furthermore, the pattern of responses seen depends not only on the pattern of isozymes present but may also depend on the age of the animal because there was no simple relationship between the apparent sensitivity of the contractile apparatus for calcium and proportion of isozyme in the myocardium.