Differential regulation of Ca2+-activated K+ channels by beta-adrenoceptors in guinea pig urinary bladder smooth muscle

Stimulation of -adrenoceptors contributes to the relaxation of urinary bladder smooth muscle (UBSM) through activation of large-conductance Ca²⁺-activated K⁺ (BK) channels. We examined the mechanisms by which -adrenoceptor stimulation leads to an elevation of the activity of BK channels in UBSM. Depolarization from –70 to +10 mV evokes an inward L-type dihydropyridine-sensitive voltage-dependent Ca²⁺ channel (VDCC) current, followed by outward steady-state and transient BK current. In the presence of ryanodine, which blocks the transient BK currents, isoproterenol, a nonselective -adrenoceptor agonist, increased the VDCC current by 25% and the steady-state BK current by 30%. In the presence of the BK channel inhibitor iberiotoxin, isoproterenol did not cause activation of the remaining steady-state K⁺ current component. Decreasing Ca²⁺ influx through VDCC by nifedipine or depolarization to +80 mV suppressed the isoproterenol-induced activation of the steady-state BK current. Unlike forskolin, isoproterenol did not change significantly the open probability of single BK channels in the absence of Ca²⁺ sparks and with VDCC inhibited by nifedipine. Isoproterenol elevated Ca²⁺ spark (local intracellular Ca²⁺ release through ryanodine receptors of the sarcoplasmic reticulum) frequency and associated transient BK currents by 1.4-fold. The data support the concept that in UBSM -adrenoceptor stimulation activates BK channels by elevating Ca²⁺ influx through VDCC and by increasing Ca²⁺ sparks, but not through a Ca²⁺-independent mechanism. This study reveals key regulatory molecular and cellular mechanisms of -adrenergic regulation of BK channels in UBSM that could provide new targets for drugs in the treatment of bladder dysfunction. Ca²⁺ sparks; voltage-dependent Ca²⁺ channel; ryanodine receptor     

Beta 3-adrenergic receptor is involved in feeding regulation in chicks

We examined whether the brain beta 3-adrenergic receptor (B3-AR) is involved in the feeding regulation of chicks. Intracerebroventricular (ICV) injection of BRL37344, a B3-AR agonist, reduced food intake of chicks under ad libitum, but not fasting, feeding conditions. The ICV injection of BRL37344 did not affect chick posture or locomotion activity suggesting that BRL37344 inhibited feeding without induction of sleep-like behavior as caused by norepinephrine. Furthermore, the rectal temperature increased following the ICV injection of BRL37344. Intraperitoneal administration of BRL37344 did not reduce food intake under ad libitum feeding condition. The present study demonstrated that the brain B3-AR is involved in the inhibition of feeding in chicks. We also suggested that activation of the brain affects the energy metabolism in chicks.     

Do β3-adrenergic receptors play a role in guinea pig detrusor smooth muscle excitability and contractility?

In many species, β3-adrenergic receptors (β3-ARs) have been reported to play a primary role in pharmacologically induced detrusor smooth muscle (DSM) relaxation. However, their role in guinea pig DSM remains controversial. The aim of this study was to investigate whether β3-ARs are expressed in guinea pig DSM and to evaluate how BRL37344 and L-755,507, two selective β3-AR agonists, modulate guinea pig DSM excitability and contractility. We used a combined experimental approach including RT-PCR, patch-clamp electrophysiology, and isometric DSM tension recordings. β3-AR mRNA message was detected in freshly isolated guinea pig DSM single cells. BRL37344 but not L-755,507 caused a slight decrease in DSM spontaneous phasic contraction amplitude and frequency in a concentration-dependent manner. In the presence of atropine (1 μM), only the spontaneous phasic contractions frequency was inhibited by BRL37344 at higher concentrations. Both BRL37344 and L-755,507 significantly decreased DSM carbachol-induced phasic and tonic contractions in a concentration-dependent manner. However, only BRL37344 inhibitory effect was partially antagonized by SR59230A (10 μM), a β3-AR antagonist. In the presence of atropine, BRL37344 and L-755,507 had no inhibitory effect on electrical field stimulation-induced contractions. Patch-clamp experiments showed that BRL37344 (100 μM) did not affect the DSM cell resting membrane potential and K(+) conductance. Although β3-ARs are expressed at the mRNA level, they play a minor to no role in guinea pig DSM spontaneous contractility without affecting cell excitability. However, BRL37344 and L-755,507 have pronounced inhibitory effects on guinea pig DSM carbachol-induced contractions. The study outlines important DSM β3-ARs species differences.     

Low-affinity state beta1-adrenoceptor-induced vasodilation in SHR

Low-affinity state beta1-adrenoceptor (beta1-AR) was functionally expressed in some blood vessels and was different from beta1, beta2 and beta3-AR. In rat aorta, low-affinity state beta1-AR activation produced an endothelium-independent relaxation which was impaired in spontaneously hypertensive rats (SHRs). In the present work, we investigated whether renin-angiotensin system was involved in this alteration by evaluating the effects of enalapril, an angiotensin converting enzyme (ACE) inhibitor or losartan, an AT1 angiotensin receptor antagonist. Cumulative concentration-response curves to low-affinity state beta1-AR agonists (CGP 12177, cyanopindolol or alprenolol) and to NS 1619, a large conductance Ca2+-activated K+ channels (BK) agonist were performed in denuded aortic rings isolated from control or treated Wistar Kyoto (WKY) rats or SHRs in different experimental conditions. The low-affinity state beta1-AR-mediated aortic vasodilation was impaired in 5 and 12 weeks old SHRs when compared to age-matched WKY. Twelve days enalapril (5 mg/kg/day) or losartan (15 mg/kg/day) treatments reduced systolic blood pressure (SBP) only in 12 weeks old SHRs whereas no significant change was observed in other groups. These treatments improved low-affinity state beta1-AR effect only in SHRs groups. In 12 weeks old WKY rats, CGP 12177-induced relaxation was insensitive to glibenclamide, a K(ATP)+ channel blocker, but was reduced by TEA or iberiotoxin, two large conductance Ca2+-activated K+ channel (BK) blockers. The impairment of NS 1619-induced vasodilation in both 5 and 12 weeks old SHRs was restored by enalapril or losartan. These results suggested that improvement of the low-affinity state beta1-AR-mediated vasodilation in 5 and 12 weeks old SHRs could be attributed to enhanced BK channels-induced hyperpolarization in SHRs independently of lowering of SBP.     

Coexistence of at least three distinct beta-adrenoceptors in human internal mammary artery.

The internal mammary artery (IMA) is currently the preferred conduit for myocardial revascularization. However, perioperative vasospasm and a hypoperfusion state during maximal exercise may limit its use as a bypass graft. The mechanism of spasm has not been clearly defined. Since beta-adrenoceptor activation plays a major role in vasorelaxation, the present study was carried out to investigate the beta-adrenoceptor responsiveness of human IMA smooth muscle. Isoproterenol produced a concentration-dependent relaxation in endothelium-denuded IMA segments, precontracted with phenylephrine (maximal relaxation 46.33+/-5.45%). Atenolol (10(-6)M) and propranolol (2x10(-7)M) inhibited isoproterenol-induced relaxation. While atenolol produced partial inhibition, propranolol caused a complete inhibition in a majority of the segments and a partial inhibition in a minority. BRL 37344, a selective beta 3-adrenoceptor agonist, produced a concentration-dependent relaxation in phenylephrine-precontracted rings of endothelium-denuded IMA (maximal relaxation 40.35+/-4.07%). Cyanopindolol, a beta-adrenoceptor partial agonist, produced a marked relaxation (58.65+/-6.2%) in endothelium-denuded IMA rings, precontracted with phenylephrine. Cyanopindolol-induced relaxation was resistant to blockade by propranolol (2x10(-7)M). Spontaneous contractions of IMA rings were also observed in some cases that were inhibited by isoproterenol and BRL 37344. This observation implies the important role of beta-adrenoceptor activation in prevention of human IMA spasm. The results obtained in present study indicate that human IMA smooth muscle possesses an atypical beta-adrenoceptor together with beta1- and beta2-adrenoceptors. Regarding the relaxation induced in IMA rings by adding BRL 37344, the possible identical entities of IMA atypical beta-adrenoceptors and beta 3-adrenoceptors are suggested.     

EETs relax airway smooth muscle via an EpDHF effect: BK(Ca) channel activation and hyperpolarization

Epoxyeicosatrienoic acids (EETs) are produced from arachidonic acid via the cytochrome P-450 epoxygenase pathway. EETs are able to modulate smooth muscle tone by increasing K(+) conductance, hence generating hyperpolarization of the tissues. However, the molecular mechanisms by which EETs induce smooth muscle relaxation are not fully understood. In the present study, the effects of EETs on airway smooth muscle (ASM) were investigated using three electrophysiological techniques. 8,9-EET and 14,15-EET induced concentration-dependent relaxations of the ASM precontracted with a muscarinc agonist (carbamylcholine chloride), and these relaxations were partly inhibited by 10 nM iberiotoxin (IbTX), a specific large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel blocker. Moreover, 3 microM 8,9- or 14,15-EET induced hyperpolarizations of -12 +/- 3.5 and -16 +/- 3 mV, with EC(50) values of 0.13 and 0.14 microM, respectively, which were either reversed or blocked on addition of 10 nM IbTX. These results indicate that BK(Ca) channels are involved in hyperpolarization and participate in the relaxation of ASM. In addition, complementary experiments demonstrated that 8,9- and 14,15-EET activate reconstituted BK(Ca) channels at low free Ca(2+) concentrations without affecting their unitary conductance. These increases in channel activity were IbTX sensitive and correlated well with the IbTX-sensitive hyperpolarization and relaxation of ASM. Together these results support the view that, in ASM, the EETs act through an epithelium-derived hyperpolarizing factorlike effect.     

Atypical beta-adrenergic receptor in 3T3-F442A adipocytes. Pharmacological and molecular relationship with the human beta 3-adrenergic receptor.

Expression of ligand binding properties for an atypical beta-adrenergic receptor (beta-AR) subtype was studied during the adipose differentiation of murine 3T3-F442A cells and compared with that of the human beta 3-AR expressed in Chinese hamster ovary cells stably transfected with the human beta 3-AR gene (CHO-beta 3 cells) Emorine, L. J., Marullo, S., Briend-Sutren, M. M., Patey, G., Tate, K., Delavier-Klutchko, C., and Strosberg, A. D. (1989) Science 245, 1118-1121). 3T3-F442A adipocytes exhibited high and low affinity binding sites for (-)-4-(3-t-butylamino-2-hydroxypropoxy) [5,7-3H]benzimidazole-2-one ((-)-[3H]CGP-12177) (KD = 1.2 and 38.3 nM) and (-)-[125I]iodocyanopindolol ([125I]CYP) (KD = 47 and 1,510 pM). The high affinity sites corresponded to the classical beta 1- and beta 2-AR subtypes whereas the KD values of the low affinity sites for the radioligands were similar to those measured in CHO-beta 3 cells (KD = 28 nM and 1,890 pM for (-)-[3H]CGP12177 and [125I]CYP, respectively). These low affinity sites were undetectable in preadipocytes but represented about 90% of total beta-ARs in adipocytes. The atypical beta-AR and the human beta 3-AR add similarly low affinities (Ki = 3-5 microM) for (+/-)-(2-(3-carbamoyl-4-hydroxyphenoxy)ethylamino-3)-(4-(1-methyl- 4- trifluormethyl-2-imidazolyl)-phenoxy)-2-propanol methane sulfonate (CGP20712A) or erythro-(+/-)-1-(7-methylindan-4-yloxy)-3-isopropylaminob utan-2-ol (ICI118551), highly selective beta 1- and beta 2-AR antagonists, respectively, in agreement with the poor inhibitory effect of the compounds on (-)-isoproterenol (IPR)-stimulated adenylate cyclase activity. Atypical beta-AR and beta 3-AR had an affinity about 10-50 times higher for sodium-4-(2-[2-hydroxy-2-(3-chlorophenyl)ethylamino]propyl)phenoxyace tate sesquihydrate (BRL37344) than the beta 1-AR subtype. This correlates with the potent lipolytic effect of BRL37344 in adipocytes. The rank order of potency of agonists in functional and binding studies was BRL37344 greater than IPR less than (-)-norepinephrine greater than (-)-epinephrine both in 3T3 adipocytes and CHO-beta 3 cells. As in CHO-beta 3 cells, the classical beta 1- and beta 2-antagonists CGP12177, oxprenolol, and pindolol were partial agonists in adipocytes. Although undetectable in preadipocytes, a major mRNA species of 2.3 kilobases (kb) and a minor one of 2.8 kb were observed in adipocytes by hybridization to a human beta 3-AR specific probe.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of the beta(3)-adrenergic receptor and/or a putative beta(4)-adrenergic receptor on the expression of uncoupling proteins and peroxisome proliferator-activated receptor-gamma coactivator-1.

Administration of beta-adrenergic receptor (beta-AR) agonists, especially beta(3)-AR agonists, is well known to increase thermogenesis in rodents and humans. In this work we studied the role of the beta(3)-AR in regulating mRNA expression of genes involved in thermogenesis, i.e., mitochondrial uncoupling proteins UCP2 and UCP3, and peroxisome proliferator-activated receptor-gamma coactivator-1 (PGC-1), in mouse skeletal muscle. For this purpose, different beta(3)-AR agonists were administered acutely to both wild type mice and mice whose beta(3)-AR gene has been disrupted (beta(3)-AR KO mice). CL 316243 increased the expression of UCP2, UCP3 and PGC-1 in wild type mice only. By contrast, BRL 37344 and CGP 12177 increased the expression of UCP2 and UCP3 in both wild type and beta(3)-AR KO mice, whereas they increased the expression of PGC-1 in wild type mice only. Finally, acute (3 h) cold exposure increased the expression of UCP2 and UCP3, but not PGC-1, in skeletal muscle of both wild type and beta(3)-AR KO mice. These results show that selective stimulation of the beta(3)-AR affects the expression of UCP2, UCP3 and PGC-1 in skeletal muscle. This effect is probably indirect, as muscle does not seem to express beta(3)-AR. In addition, our data suggest that BRL 37344 and CGP 12177 act, in part, through an as yet unidentified receptor, possibly a beta(4)-AR.     

Regulation of Guinea Pig Detrusor Smooth Muscle Excitability by 17β-Estradiol: The Role of the Large Conductance Voltage- and Ca2+-Activated K+ Channels

Estrogen replacement therapies have been suggested to be beneficial in alleviating symptoms of overactive bladder. However, the precise regulatory mechanisms of estrogen in urinary bladder smooth muscle (UBSM) at the cellular level remain unknown. Large conductance voltage- and Ca²⁺-activated K⁺ (BK) channels, which are key regulators of UBSM function, are suggested to be non-genomic targets of estrogens. This study provides an electrophysiological investigation into the role of UBSM BK channels as direct targets for 17β-estradiol, the principle estrogen in human circulation. Single BK channel recordings on inside-out excised membrane patches and perforated whole cell patch-clamp were applied in combination with the BK channel selective inhibitor paxilline to elucidate the mechanism of regulation of BK channel activity by 17β-estradiol in freshly-isolated guinea pig UBSM cells. 17β-Estradiol (100 nM) significantly increased the amplitude of depolarization-induced whole cell steady-state BK currents and the frequency of spontaneous transient BK currents in freshly-isolated UBSM cells. The increase in whole cell BK currents by 17β-estradiol was eliminated upon blocking BK channels with paxilline. 17β-Estradiol (100 nM) significantly increased (~3-fold) the single BK channel open probability, indicating direct 17β-estradiol-BK channel interactions. 17β-Estradiol (100 nM) caused a significant hyperpolarization of the membrane potential of UBSM cells, and this hyperpolarization was reversed by blocking the BK channels with paxilline. 17β-Estradiol (100 nM) had no effects on L-type voltage-gated Ca²⁺ channel currents recorded under perforated patch-clamp conditions. This study reveals a new regulatory mechanism in the urinary bladder whereby BK channels are directly activated by 17β-estradiol to reduce UBSM cell excitability.     

Negative feedback regulation of nerve-mediated contractions by KCa channels in mouse urinary bladder smooth muscle

When the urinary bladder is full, activation of parasympathetic nerves causes release of neurotransmitters that induce forceful contraction of the detrusor muscle, leading to urine voiding. The roles of ion channels that regulate contractility of urinary bladder smooth muscle (UBSM) in response to activation of parasympathetic nerves are not well known. The present study was designed to characterize the role of large (BK)- and small-conductance (SK) Ca(2+)-activated K(+) (K(Ca)) channels in regulating UBSM contractility in response to physiological levels of nerve stimulation in UBSM strips from mice. Nerve-evoked contractions were induced by electric field stimulation (0.5-50 Hz) in isolated strips of UBSM. BK and SK channel inhibition substantially increased the amplitude of nerve-evoked contractions up to 2.45 +/- 0.12- and 2.99 +/- 0.25-fold, respectively. When both SK and BK channels were inhibited, the combined response was additive. Inhibition of L-type voltage-dependent Ca(2+) channels (VDCCs) in UBSM inhibited nerve-evoked contractions by 92.3 +/- 2.0%. These results suggest that SK and BK channels are part of two distinct negative feedback pathways that limit UBSM contractility in response to nerve stimulation by modulating the activity of VDCCs. Dysfunctional regulation of UBSM contractility by alterations in BK/SK channel expression or function may underlie pathologies such as overactive bladder.     

Spontaneous transient outward currents arise from microdomains where BK channels are exposed to a mean Ca(2+) concentration on the order of 10 microM during a Ca(2+) spark

Ca(2+) sparks are small, localized cytosolic Ca(2+) transients due to Ca(2+) release from sarcoplasmic reticulum through ryanodine receptors. In smooth muscle, Ca(2+) sparks activate large conductance Ca(2+)-activated K(+) channels (BK channels) in the spark microdomain, thus generating spontaneous transient outward currents (STOCs). The purpose of the present study is to determine experimentally the level of Ca(2+) to which the BK channels are exposed during a spark. Using tight seal, whole-cell recording, we have analyzed the voltage-dependence of the STOC conductance (g((STOC))), and compared it to the voltage-dependence of BK channel activation in excised patches in the presence of different [Ca(2+)]s. The Ca(2+) sparks did not change in amplitude over the range of potentials of interest. In contrast, the magnitude of g((STOC)) remained roughly constant from 20 to -40 mV and then declined steeply at more negative potentials. From this and the voltage dependence of BK channel activation, we conclude that the BK channels underlying STOCs are exposed to a mean [Ca(2+)] on the order of 10 microM during a Ca(2+) spark. The membrane area over which a concentration > or =10 microM is reached has an estimated radius of 150-300 nm, corresponding to an area which is a fraction of one square micron. Moreover, given the constraints imposed by the estimated channel density and the Ca(2+) current during a spark, the BK channels do not appear to be uniformly distributed over the membrane but instead are found at higher density at the spark site.     

常氧和急性低氧下大鼠传导性肺动脉平滑肌细胞的电生理特性

本文旨在研究大鼠传导性肺动脉平滑肌细胞（pulmonary artery smooth muscle cells,PASMCs）的电生理特征及对急性低氧的反应。用酶解法急性分离出1-2级分支的PASMCs,通过全细胞膜片钳方法研究常氧及急性低氧状况下细胞钾电流的差异,并在常氧下先后使用iBTX和4-AP阻断大电导钙激活钾离子（large conductance Ca-activated K^＋,BKCa）通道及延迟整流性钾离子（delayed rectifier K^＋,KDR）通道后,观察细胞钾电流特征。根据细胞的大小、形态及电生理特征可将PASMCs分为Ⅰ、Ⅱ、Ⅲ类。iBTX对Ⅰ类细胞几乎无作用,而4-AP几乎完全阻断它的钾电流;Ⅱ类细胞的钾电流在加入iBTX后大部分被抑制,其余的对4．AP敏感;Ⅲ类细胞的钾电流对iBTX及4-AP均敏感。急性低氧对三类细胞的钾电流均有不同程度的抑制,并使Ⅰ类细胞的膜电位显著升高,而Ⅱ、Ⅲ类细胞膜电位升高的程度不如Ⅰ类显著。结果表明,传导性肺动脉有3种形态及电生理特性不同的PASMCs,在急性低氧时其钾电流不同程度地受到抑制,同时静息膜电位也有不同程度去极化,这些可能参与急性低氧时传导性肺动脉舒缩反应的调节。KDR及BKCa通道在3种细胞中的比例不同可能是急性低氧对3种PASMCs影响不同的离子基础。     

Properties of a new calcium-permeable single channel from tracheal microsomes

After the incorporation of the tracheal microsomal membrane into bilayer lipid membrane (BLM), a new single channel permeable for calcium was observed. Using the BLM conditions, 53 mM Ca2+ in trans solution versus 200 nM Ca2+ in cis solution, the single calcium channel current at 0 mV was 1.4-2.1 pA and conductance was 62-75 pS. The channel Ca2+/K+ permeability ratio was 4.8. The open probability (P-open) was in the range of 0.7-0.97. The P-open, measured at -10 mV to +30 mV (trans-cis), was not voltage dependent. The channel was neither inhibited by 10-20 microM ruthenium red, a specific blocker of ryanodine calcium release channel, nor by 10-50 microM heparin, a specific blocker of IP3 receptor calcium release channel, and its activity was not influenced by addition of 0.1 mM MgATP. We suggest that the observed new channel is permeable for calcium, and it is neither identical with the known type 1 or 2 ryanodine calcium release channel, nor type 1 or 2 IP3 receptor calcium release channel.     

Frequency encoding of cholinergic- and purinergic-mediated signaling to mouse urinary bladder smooth muscle: modulation by BK channels

In the urinary bladder, contractions of the detrusor muscle and urine voiding are induced by the neurotransmitters ACh and ATP, released from parasympathetic nerves. Activation of K(+) channels, in particular the large-conductance Ca(2+)-activated K(+) (BK) channels, opposes increases in excitability and contractility of urinary bladder smooth muscle (UBSM). We have shown that deleting the gene mSlo1 in mice (Slo(-/-)), encoding the BK channel, leads to enhanced nerve-mediated and neurotransmitter-dependent contractility of UBSM (38). Here, we examine the location of the BK channel in urinary bladder strips from mouse. Immunohistochemical analysis revealed that the channel is expressed in UBSM but not in nerves that innervate the smooth muscle. The relationship between electrical field stimulation and force generation of the cholinergic and purinergic pathways was examined by applying blockers of the respective receptors in UBSM strips from wild-type and from Slo(-/-) (knockout) mice. In wild-type strips, the stimulation frequency required to obtain a half-maximal force was significantly lower for the purinergic (7.2 +/- 0.3 Hz) than the cholinergic pathway (19.1 +/- 1.5 Hz), whereas the maximum force was similar. Blocking BK channels with iberiotoxin or ablation of the Slo gene increased cholinergic- and purinergic-mediated force at low frequencies, i.e., significantly decreased the frequency for a half-maximal force. Our results indicate that the BK channel has a very significant role in reducing both cholinergic- and purinergic-induced contractility and suggest that alterations in BK channel expression or function could contribute to pathologies such as overactive detrusor.     

Functional alterations in cerebrovascular K(+) and Ca(2+) channels are comparable between simulated microgravity rat and SHR

Exposure to microgravity leads to a sustained elevation in transmural pressure across the cerebral vasculature due to removal of hydrostatic pressure gradients. We hypothesized that ion channel remodeling in cerebral vascular smooth muscle cells (VSMCs) similar to that associated with hypertension may occur and play a role in upward autoregulation of cerebral vessels during microgravity. Sprague-Dawley rats were subjected to 4-wk tail suspension (Sus) to simulate the cardiovascular effect of microgravity. Large-conductance Ca(2+)-activated K(+) (BK(Ca)), voltage-gated K(+) (K(V)), and L-type voltage-dependent Ca(2+) (Ca(L)) currents of Sus and control (Con) rat cerebral VSMCs were investigated with a whole cell voltage-clamp technique. Under the same experimental conditions, K(V), BK(Ca), and Ca(L) currents of cerebral VSMCs from adult spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) were also investigated. K(V) current density decreased in Sus rats vs. Con rats [1.07 +/- 0.14 (n = 22) vs. 1.31 +/- 0.28 (n = 16) pA/pF at +20 mV (P < 0.05)] and BK(Ca) and Ca(L) current densities increased [BK(Ca): 1.70 +/- 0.37 (n = 23) vs. 0.88 +/- 0.22 (n = 19) pA/pF at +20 mV (P < 0.05); Ca(L): -2.17 +/- 0.21 (n = 35) vs. -1.31 +/- 0.10 (n = 26) pA/pF at +10 mV (P < 0.05)]. Similar changes were also observed in SHR vs. WKY cerebral VSMCs: K(V) current density decreased [1.03 +/- 0.33 (n = 9) vs. 1.62 +/- 0.64 (n = 9) pA/pF at +20 mV (P < 0.05)] and BK(Ca) and Ca(L) current densities increased [BK(Ca): 2.54 +/- 0.47 (n = 11) vs. 1.12 +/- 0.33 (n = 12) pA/pF at +20 mV (P < 0.05); Ca(L): -3.99 +/- 0.53 (n = 12) vs. -2.28 +/- 0.20 (n = 10) pA/pF at +20 mV (P < 0.05)]. These findings support our hypothesis, and their impact on space cardiovascular research is discussed.     

Reduced functional expression of K(+) channels in vascular smooth muscle cells from rats made hypertensive with N{omega}-nitro-L-arginine

Smooth muscle membrane potential is determined, in part, by K(+) channels. In the companion paper to this article, we demonstrated that superior mesenteric arteries from rats made hypertensive with N(omega)-nitro-l-arginine (l-NNA) are depolarized and express less K(+) channel protein compared with those from normotensive rats. In the present study, we used patch-clamp techniques to test the hypothesis that l-NNA-induced hypertension reduces the functional expression of K(+) channels in smooth muscle. In whole cell experiments using a Ca(2+)-free pipette solution, current at 0 mV, largely due to voltage-dependent K(+) (K(V)) channels, was reduced approximately 60% by hypertension (2.7 +/- 0.4 vs. 1.1 +/- 0.2 pA/pF). Current at +100 mV with 300 nM free Ca(2+), largely due to large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels, was reduced approximately 40% by hypertension (181 +/- 24 vs. 101 +/- 28 pA/pF). Current blocked by 3 mM 4-aminopyridine, an inhibitor of many K(V) channel types, was reduced approximately 50% by hypertension (1.0 +/- 0.4 vs. 0.5 +/- 0.2 pA/pF). Current blocked by 1 mM tetraethylammonium, an inhibitor of BK(Ca) channels, was reduced approximately 40% by hypertension (86 +/- 14 vs. 53 +/- 19 pA/pF). Differences in BK(Ca) current magnitude are not attributable to changes in single-channel conductance or Ca(2+)/voltage sensitivity. The data support the hypothesis that l-NNA-induced hypertension reduces K(+) current in vascular smooth muscle. Reduced molecular and functional expression of K(+) channels may partly explain the depolarization and augmented contractile sensitivity of smooth muscle from l-NNA-treated rats.     

Whole cell current and membrane potential regulation by a human smooth muscle mechanosensitive calcium channel

Mechanotransduction is required for a wide variety of biological functions. The aim of this study was to determine the effect of activation of a mechanosensitive Ca(2+) channel, present in human jejunal circular smooth muscle cells, on whole cell currents and on membrane potential. Currents were recorded using patch-clamp techniques, and perfusion of the bath (10 ml/min, 30 s) was used to mechanoactivate the L-type Ca(2+) channel. Perfusion resulted in activation of L-type Ca(2+) channels and an increase in outward current from 664 +/- 57 to 773 +/- 72 pA at +60 mV. Membrane potential hyperpolarized from -42 +/- 4 to -50 +/- 5 mV. In the presence of nifedipine (10 microM), there was no increase in outward current or change in membrane potential with perfusion. In the presence of charybdotoxin or iberiotoxin, perfusion of the bath did not increase outward current or change membrane potential. A model is proposed in which mechanoactivation of an L-type Ca(2+) channel current in human jejunal circular smooth muscle cells results in increased Ca(2+) entry and cell contraction. Ca(2+) entry activates large-conductance Ca(2+)-activated K(+) channels, resulting in membrane hyperpolarization and relaxation.     

Regulation of urinary bladder smooth muscle contractions by ryanodine receptors and BK and SK channels

This study examines the roles of voltage-dependent Ca(2+) channels (VDCC), ryanodine receptors (RyRs), large-conductance Ca(2+)-activated K(+) (BK) channels, and small-conductance Ca(2+)-activated K(+) (SK) channels in the regulation of phasic contractions of guinea pig urinary bladder smooth muscle (UBSM). Nisoldipine (100 nM), a dihydropyridine inhibitor of VDCC, abolished spontaneous UBSM contractions. Ryanodine (10 microM) increased contraction frequency and thereby integrated force and, in the presence of the SK blocker apamin, had a greater effect on integrated force than ryanodine alone. Blocking BK (iberiotoxin, 100 nM) or SK (apamin, 100 nM) channels increased contraction amplitude and duration but decreased frequency. The contractile response to iberiotoxin was more pronounced than to apamin. The increases in contraction amplitude and duration to apamin were substantially augmented with ryanodine pretreatment. These results indicate that BK and SK channels have prominent roles as negative feedback elements to limit UBSM contraction amplitude and duration. RyRs also appear to play a significant role as a negative feedback regulator of contraction frequency and duration, and this role is influenced by the activity of SK channels.