Opposing roles of K(+) and Cl(-) channels in maintenance of opossum lower esophageal sphincter tone

The ionic basis underlying the maintenance of myogenic tone of lower esophageal sphincter circular muscle (LES) was investigated in opossum with the use of standard isometric tension and conventional intracellular microelectrode recordings in vitro. In tension recording studies, nifedipine (1 microM) reduced basal tone to 27.7 +/- 3.8% of control. The K(+) channel blockers tetraethylammonium (TEA, 2 mM), charybdotoxin (100 nM), and 4-aminopyridine (4-AP, 2 mM) enhanced resting tone, whereas apamin and glibenclamide were without affect. Cl(-) channel blockers DIDS (500 microM) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (500 microM), as well as niflumic acid (0.1-300 microM), decreased basal tone, but tamoxifen was without effect. Intracellular microelectrode recordings revealed ongoing, spontaneous, spike-like action potentials (APs). Nifedipine abolished APs and depolarized resting membrane potential (RMP). Both TEA and 4-AP significantly depolarized RMP and augmented APs, whereas niflumic acid dose-dependently hyperpolarized RMP and abolished APs. These data suggest that, in the opossum, basal tone is associated with continuous APs and that K(+) and Ca(2+)-activated Cl(-) channels have important opposing roles in the genesis of LES tone.     

Effects of temperature on slow and fast inactivation of rat skeletal muscle Na+ channels

Patch-clampstudies of mammalian skeletal muscleNa⁺ channels are commonly done atsubphysiological temperatures, usually room temperature. However, atsubphysiological temperatures, mostNa⁺ channels are inactivated atthe cell resting potential. This study examined the effects oftemperature on fast and slow inactivation ofNa⁺ channels to determine iftemperature changed the fraction of Na⁺ channels that were excitableat resting potential. The loose patch voltage clamp recordedNa⁺ currents(I_Na) in vitroat 19, 25, 31, and 37°C from the sarcolemma of rat type IIbfast-twitch omohyoid skeletal muscle fibers. Temperature affected thefraction of Na⁺ channels that wereexcitable at the resting potential. At 19°C, only 30% of channelswere excitable at the resting potential. In contrast, at 37°C, 93%of Na⁺ channels were excitable atthe resting potential. Temperature did not alter the resting potentialor the voltage dependencies of activation or fast inactivation.I_Na available atthe resting potential increased with temperature because thesteady-state voltage dependence of slow inactivation shifted in adepolarizing direction with increasing temperature. The membranepotential at which half of the Na⁺channels were in the slow inactivated state was shifted by +16 mV at37°C compared with 19°C. Consequently, the low availability ofexcitable Na⁺ channels atsubphysiological temperatures resulted from channels being in the slow,inactivated state at the resting potential.

     

Apoptosis repressor with caspase domain inhibits cardiomyocyte apoptosis by reducing K+ currents

Cell shrinkageis an early prerequisite in programmed cell death, and cytoplasmicK⁺ is a dominant cation that controls intracellular ionhomeostasis and cell volume. Blockade of K⁺ channelsinhibits apoptotic cell shrinkage and attenuates apoptosis. We examined whether apoptotic repressor with caspase recruitment domain (ARC), an antiapoptotic protein, inhibits cardiomyocyte apoptosis by reducing K⁺ efflux throughvoltage-gated K⁺ (Kv) channels. In heart-derived H9c2cells, whole cell Kv currents (I_K(V)) wereisolated by using Ca²⁺-free extracellular (bath) solutionand including 5 mM ATP and 10 mM EGTA in the intracellular (pipette)solution. Extracellular application of 5 mM 4-aminopyridine (4-AP), ablocker of Kv channels, reversibly reduced I_K(V)by 50-60% in H9c2 cells. The remaining currents during 4-APtreatment may be generated by K⁺ efflux through4-AP-insensitive K⁺ channels. Overexpression of ARC inheart-derived H9c2 cells significantly decreasedI_K(V), whereas treatment with staurosporine, apotent apoptosis inducer, enhanced I_K(V)in wild-type cells. The staurosporine-induced increase inI_K(V) was significantly suppressed and thestaurosporine-mediated apoptosis was markedly inhibited incells overexpressing ARC compared with cells transfected with thecontrol neomycin vector. These results suggest that theantiapoptotic effect of ARC is, in part, due to inhibition of Kvchannels in cardiomyocytes.

     

Gender-specific K(+)-channel contribution to adenosine-induced relaxation in coronary arterioles.

We examined the contribution of K(+)-channel activity on basal tone and adenosine-mediated relaxation of coronary arterioles isolated from sexually mature male and female miniature swine. Arterioles (approximately 100-200 microm ID) isolated from the apical region of the heart were cannulated and studied using videodimensional analysis under constant intraluminal pressure. Coronary arterioles from male and female pigs demonstrated similar levels of basal tone and reductions in basal diameter in response to the K(+)-channel blockers 4-aminopyridine (4-AP; 1 mM), tetraethylammonium (1 mM), and glibenclamide (Glib; 10 microM), with 4-AP producing significantly greater constriction than tetraethylammonium or Glib. After endothelin-induced preconstriction, relaxation responses to adenosine were not significantly different between coronary arterioles of male and female pigs. Inhibition of 4-AP-sensitive channels significantly impaired adenosine-mediated relaxation in arterioles from male but not female pigs. However, inhibition of K(+) channels with iberiotoxin (100 nM) or Glib had no effect on adenosine-induced relaxation in either sex. Results obtained in the presence of nitric oxide synthase inhibition suggest a potential interaction of 4-AP-sensitive channels and nitric oxide at low adenosine concentrations. In conclusion, our data indicate that 4-AP-sensitive channels 1) contribute significantly to basal tone in coronary arterioles of both male and female pigs, 2) contribute to adenosine-mediated relaxation in male but not female pigs, and 3) can contribute to adenosine-induced relaxation independent of nitric oxide production in male pigs. These data are consistent with a significant role for voltage-dependent K(+) channels in adenosine-mediated relaxation of coronary arterioles from males.     

Short-term exercise enhances insulin-stimulated GLUT-4 translocation and glucose transport in adipose cells

This investigation examined the effectsof short-term exercise training on insulin-stimulated GLUT-4 glucosetransporter translocation and glucose transport activity in rat adiposecells. Male Wistar rats were randomly assigned to a sedentary (Sed) orswim training group (Sw, 4 days; final 3 days: 2 × 3 h/day). Adipose cell size decreased significantly but minimally(~20%), whereas total GLUT-4 increased by 30% in Sw vs. Sed rats.Basal3-O-methyl-D-[¹⁴C]glucosetransport was reduced by 62%, whereas maximally insulin-stimulated (MIS) glucose transport was increased by 36% in Sw vs. Sed rats. MIScell surface GLUT-4 photolabeling was 44% higher in the Sw vs. Sedanimals, similar to the increases observed in MIS glucose transportactivity and total GLUT-4. These results suggest that increases intotal GLUT-4 and GLUT-4 translocation to the cell surface contribute tothe increase in MIS glucose transport with short-term exercisetraining. In addition, the results suggest that the exercisetraining-induced adaptations in glucose transport occur more rapidlythan previously thought and with minimal changes in adipose cell size.

     

Characterization of outward K(+) currents in isolated smooth muscle cells from sheep urethra

The perforated-patch techniquewas used to measure membrane currents in smooth muscle cells from sheepurethra. Depolarizing pulses evoked large transient outward currentsand several components of sustained current. The transient current anda component of sustained current were blocked by iberiotoxin, penitremA, and nifedipine but were unaffected by apamin or 4-aminopyridine,suggesting that they were mediated by large-conductanceCa²⁺-activated K⁺ (BK) channels. When the BKcurrent was blocked by exposure to penitrem A (100 nM) andCa²⁺-free bath solution, there remained a voltage-sensitiveK⁺ current that was moderately sensitive to blockade withtetraethylammonium (TEA; half-maximal effective dose = 3.0 ± 0.8 mM) but not 4-aminopyridine. Penitrem A (100 nM) increasedthe spike amplitude and plateau potential in slow waves evoked insingle cells, whereas addition of TEA (10 mM) further increased theplateau potential and duration. In conclusion, bothCa²⁺-activated and voltage-dependent K⁺currents were found in urethral myocytes. Both of these currents arecapable of contributing to the slow wave in these cells, suggesting that they are likely to influence urethral tone under certain conditions.

     

Modulation of NK cell cytolytic activity by macrophages in chronically exercise-stressed mice

Blank, Sally E., T. Bucky Jones, Eric G. Lee, C. JayneBrahler, Randle M. Gallucci, Marne L. Fox, and Gary G. Meadows. Modulation of NK cell cytolytic activity by macrophages in chronically exercise-stressed mice. J. Appl.Physiol. 83(3): 845-850, 1997.This study wasdesigned to investigate the effects of moderate-intensity endurancetraining on basal natural killer (NK) cell cytolytic activity in murinesplenocytes that were enriched for1)NK1.1⁺ cells or2) macrophages andNK1.1⁺ cells. Mice were assignedto sedentary (Sed), treadmill control (TM), or treadmill-trained (Trn)groups. Splenocyte number, the percentages ofNK1.1⁺, large granular lymphocytes(NK1.1⁺, LGL-1⁺),and other subpopulations did not change in Trn mice. Approximately 70%of cells enriched for NK1.1⁺expressed this surface antigen. Lytic units (LU) expressed per LGL-1⁺ cell were significantlylower in Trn [83.9 ± 3.2 (SE)] compared with Sed (109.5 ± 7.5) and TM (101.3 ± 6.4) groups. When macrophages remainedin the in vitro assay, LU perLGL-1⁺ cell did not differ acrossgroups. The results indicate that highly enrichedNK1.1⁺ cells from Trn mice hadlower NK cell activity compared with Sed mice. No differences in NKcell activity were observed when cells were enriched forNK1.1⁺ cells and macrophages.These findings support the hypothesis that macrophage modulation of NKcells may be one mechanism contributing to augmented basal NK cellactivity in endurance-trained individuals.

     

Na+K+-ATPase Activity and K+ Channels Differently Contribute to Vascular Relaxation in Male and Female Rats

Gender associated differences in vascular reactivity regulation might contribute to the low incidence of cardiovascular disease in women. Cardiovascular protection is suggested to depend on female sex hormones’ effects on endothelial function and vascular tone regulation. We tested the hypothesis that potassium (K⁺) channels and Na⁺K⁺-ATPase may be involved in the gender-based vascular reactivity differences. Aortic rings from female and male rats were used to examine the involvement of K⁺ channels and Na⁺K⁺-ATPase in vascular reactivity. Acetylcholine (ACh)-induced relaxation was analyzed in the presence of L-NAME (100 µM) and the following K⁺ channels blockers: tetraethylammonium (TEA, 2 mM), 4-aminopyridine (4-AP, 5 mM), iberiotoxin (IbTX, 30 nM), apamin (0.5 µM) and charybdotoxin (ChTX, 0.1 µM). The ACh-induced relaxation sensitivity was greater in the female group. After incubation with 4-AP the ACh-dependent relaxation was reduced in both groups. However, the dAUC was greater in males, suggesting that the voltage-dependent K⁺ channel (K_v) participates more in males. Inhibition of the three types of Ca²⁺-activated K⁺ channels induced a greater reduction in R_max in females than in males. The functional activity of the Na⁺K⁺-ATPase was evaluated by KCl-induced relaxation after L-NAME and OUAincubation. OUA reduced K⁺-induced relaxation in female and male groups, however, it was greater in males, suggesting a greater Na⁺K⁺-ATPase functional activity. L-NAME reduced K⁺-induced relaxation only in the female group, suggesting that nitric oxide (NO) participates more in their functional Na⁺K⁺-ATPase activity. These results suggest that the K⁺ channels involved in the gender-based vascular relaxation differences are the large conductance Ca²⁺-activated K⁺ channels (BK_Ca) in females and K_v in males and in the K⁺-induced relaxation and the Na⁺K⁺-ATPase vascular functional activity is greater in males.     

Role of extracellular [Ca2+] in fatigue of isolated mammalian skeletal muscle

The possiblerole of altered extracellular Ca²⁺concentration([Ca²⁺]_o)in skeletal muscle fatigue was tested on isolated slow-twitch soleusand fast-twitch extensor digitorum longus muscles of the mouse. Thefollowing findings were made. 1) Achange from the control solution (1.3 mM[Ca²⁺]_o)to 10 mM[Ca²⁺]_o,or to nominally Ca²⁺-freesolutions, had little effect on tetanic force in nonfatigued muscle.2) Almost complete restoration oftetanic force was induced by 10 mM[Ca²⁺]_oin severely K⁺-depressed muscle(extracellular K⁺ concentration of10-12 mM). This effect was attributed to a 5-mV reversal of theK⁺-induced depolarization andsubsequent restoration of ability to generate action potentials(inferred by using the twitch force-stimulation strength relationship).3) Tetanic force depressed bylowered extracellular Na⁺concentration (40 mM) was further reduced with 10 mM[Ca²⁺]_o.4) Tetanic force loss at elevatedextracellular K⁺ concentration (8 mM) and lowered extracellular Na⁺concentration (100 mM) was partially reversed with 10 mM[Ca²⁺]_oor markedly exacerbated with low[Ca²⁺]_o.5) Fatigue induced by using repeatedtetani in soleus was attenuated at 10 mM[Ca²⁺]_o(due to increased resting and evoked forces) and exacerbated at low[Ca²⁺]_o.These combined results suggest, first, that raised[Ca²⁺]_oprotects against fatigue rather than inducing it and, second, that aconsiderable depletion of[Ca²⁺]_oin the transverse tubules may contribute to fatigue.

     

Effects of vigorous exercise training and beta -agonist administration on bone response to hindlimb suspension

Bloomfield, Susan A., Beverly E. Girten, and Steven E. Weisbrode. Effects of vigorous exercise training and -agonist administration on bone response to hindlimb suspension.J. Appl. Physiol. 83(1):172-178, 1997.The effectiveness of dobutamine (Dob) inpreventing bone loss during 14 days of hindlimb suspension (Sus) wastested in exercise-trained (Ex; n = 25) and sedentary (Sed; n = 22) rats(age 155 days). One-half of each group was given Dob (2 mg · kg¹ · day¹)or saline (Sal). Histomorphometric measurements at midfemur revealed a17% smaller cortical bone area (CBA) and a 32% lower periostealmineral apposition rate (MAR) in suspended vs. nonsuspended Sed/Salrats. Dob abolished this decline in CBA in Sed/Sus rats, probably via an attenuation of the decrease in periosteal MAR; similarbut nonsignificant effects on cross-sectional moment of inertia wereobserved. Nonsuspended Ex rats had no change in bone CBA when CBA isindexed to body weight. Sus appeared to uncouple the relationshipbetween soleus weight and CBA. Dob attenuated the 43% decline insoleus weight after Sus in Ex but not in Sed rats. In summary, vigorousEx before Sus does not affect loss of bone mass due to unloading; Dobeffectively maintains CBA in Sed rats subjected to suspension.

     

Activation of K+ channels induces apoptosis in vascular smooth muscle cells

Intracellular K⁺ playsan important role in controlling the cytoplasmic ion homeostasis formaintaining cell volume and inhibiting apoptotic enzymes in thecytosol and nucleus. Cytoplasmic K⁺ concentration is mainlyregulated by K⁺ uptake viaNa⁺-K⁺-ATPase and K⁺ efflux throughK⁺ channels in the plasma membrane. Carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP), a protonophorethat dissipates the H⁺ gradient across the inner membraneof mitochondria, induces apoptosis in many cell types. In ratand human pulmonary artery smooth muscle cells (PASMC), FCCP opened thelarge-conductance, voltage- and Ca²⁺-sensitiveK⁺ (maxi-K) channels, increased K⁺ currentsthrough maxi-K channels [I_K(Ca)], and inducedapoptosis. Tetraethylammonia (1 mM) and iberiotoxin (100 nM)decreased I_K(Ca) by blocking the sarcolemmalmaxi-K channels and inhibited the FCCP-induced apoptosis inPASMC cultured in media containing serum and growth factors.Furthermore, inhibition of K⁺ efflux by raisingextracellular K⁺ concentration from 5 to 40 mM alsoattenuated PASMC apoptosis induced by FCCP and theK⁺ ionophore valinomycin. These results suggest thatFCCP-mediated apoptosis in PASMC is partially due to anincrease of maxi-K channel activity. The resultant K⁺ lossthrough opened maxi-K channels may serve as a trigger for cellshrinkage and caspase activation, which are major characteristics ofapoptosis in pulmonary vascular smooth muscle cells.

     

Vasoconstrictor responses of coronary resistance arteries in exercise-trained pigs

Coronaryresistance arteries isolated from exercise-trained pigs have been shownto exhibit enhanced myogenic reactivity (J. M. Muller, P. R. Myers, andM. Harold Laughlin. J. Appl. Physiol. 75: 2677-2682, 1993). The purpose of this study was to test the hypothesis that exercise training results in enhanced vasoconstrictor responses of these arteries to all vasoconstrictor stimuli[specifically acetylcholine (ACh), endothelin-1 (ET-1), KCl, andthe Ca²⁺ channel-agonist Bay K8644]. Female Yucatan miniature swine were trained (Trn) on amotor-driven treadmill (n = 16) orremained sedentary (Sed, n = 15) for16-20 wk. Arteries 50-120 µm in diameter were isolated andcannulated with micropipettes, and intraluminal pressure was set at 60 cmH₂O throughout experiments.Vasoreactivity was evaluated by examining constrictor responses toincreasing concentrations of ACh(10⁹ to10⁴ M), ET-1(10¹⁰ to10⁸ M), KCl (bathreplacement with isotonic physiological saline solution containing 30 or 80 mM), and Bay K 8644 (10⁹ to10⁶ M). Constricteddiameters are expressed relative to the passive diameter observed after100 µM SNP. All four constrictors produced similar decreases indiameter in arteries from both groups [ACh: 0.52 ± 0.07 (Trn)and 0.54 ± 0,06 (Sed); ET-1: 0.66 ± 0.05 (Trn) and 0.70 ± 0.07 (Sed); KCl: 0.66 ± 0.05 (Trn) and 0.70 ± 0.07 (Sed); Bay K8644: 0.86 ± 0.05 (Trn) and 0.76 ± 0.05 (Sed)]. Present results combined with previous observations indicate that exercise training does not alter vasoconstrictor responses of porcine coronary resistance arteries but specifically increases myogenic reactivity. Thus the underlying cellular mechanisms for myogenic tone are alteredby training but not receptor-mediated mechanisms (ACh and ET-1) norvoltage-gated Ca²⁺ channels (KCland Bay K 8644) in coronary resistance arteries.

     

KMUP-1 ameliorates monocrotaline-induced pulmonary arterial hypertension through the modulation of Ca2+ sensitization and K+-channel

AimsThis study investigates the actions of KMUP-1 on RhoA/Rho-kinase (ROCK)-dependent Ca²⁺ sensitization and the K⁺-channel in chronic pulmonary arterial hypertension (PAH) rats.Main methodsSprague–Dawley rats were divided into control, monocrotaline (MCT), and MCT + KMUP-1 groups. PAH was induced by a single intraperitoneal injection (i.p.) of MCT (60 mg/kg). KMUP-1 (5 mg/kg, i.p.) was administered once daily for 21 days to prevent MCT-induced PAH. All rats were sacrificed on day 22.Key findingsMCT-induced increased right ventricular systolic pressure (RVSP) and right ventricular hypertrophy were prevented by KMUP-1. In myograph experiments, KCl (80 mM), phenylephrine (10 µM) and K⁺ channel inhibitors (TEA, 10 mM; paxilline, 10 µM; 4-AP, 5 mM) induced weak PA contractions in MCT-treated rats compared to controls, but the PA reactivity was restored in MCT + KMUP-1-treated rats. By contrast, in β-escin- or α-toxin-permeabilized PAs, CaCl₂-induced (1.25 mM, pCa 5.1) contractions were stronger in MCT-treated rats, and this action was suppressed in MCT + KMUP-1-treated rats. PA relaxation in response to the ROCK inhibitor Y27632 (0.1 μM) was much higher in MCT-treated rats than in control rats. In Western blot analysis, the expression of Ca²⁺-activated K⁺ (BK_Ca) and voltage-gated K⁺ channels (Kv2.1 and Kv1.5), and ROCK II proteins was elevated in MCT-treated rats and suppressed in MCT + KMUP-1-treated rats. We suggest that MCT-treated rats upregulate K⁺-channel proteins to adapt to chronic PAH.SignificanceKMUP-1 protects against PAH and restores PA vessel tone in MCT-treated rats, attributed to alteration of Ca²⁺ sensitivity and K⁺-channel function.     

Properties of ATP-dependent K(+) channels in adrenocortical cells

Bovine adrenocortical zona fasciculata (AZF)cells express a novel ATP-dependent K⁺-permeable channel(I_AC). Whole cell and single-channel recordings were used to characterize I_AC channels withrespect to ionic selectivity, conductance, and modulation bynucleotides, inorganic phosphates, and angiotensin II (ANG II). Inoutside-out patch recordings, the activity of unitaryI_AC channels is enhanced by ATP in the patchpipette. These channels were K⁺ selective with nomeasurable Na⁺ or Ca²⁺ conductance. Insymmetrical K⁺ solutions with physiological concentrationsof divalent cations (M²⁺), I_ACchannels were outwardly rectifying with outward and inward chordconductances of 94.5 and 27.0 pS, respectively. In the absence ofM²⁺, conductance was nearly ohmic. Hydrolysis-resistantnucleotides including AMP-PNP and NaUTP were more potent than MgATP asactivators of whole cell I_AC currents. Inorganicpolytriphosphate (PPP_i) dramatically enhancedI_AC activity. In current-clamp recordings, nucleotides and PPP_i produced resting potentials in AZFcells that correlated with their effectiveness in activatingI_AC. ANG II (10 nM) inhibited whole cellI_AC currents when patch pipettes contained 5 mMMgATP but was ineffective in the presence of 5 mM NaUTP and 1 mM MgATP.Inhibition by ANG II was not reduced by selective kinase antagonists.These results demonstrate that I_AC is adistinctive K⁺-selective channel whose activity isincreased by nucleotide triphosphates and PPP_i.Furthermore, they suggest a model for I_AC gatingthat is controlled through a cycle of ATP binding and hydrolysis.

     

Desensitization to ANG II in guinea pig ileum depends on membrane repolarization: role of maxi-K+ channel

Desensitization of ANG II tonic contractile response of theguinea pig ileum is related to membrane repolarization determined byCa²⁺-activatedK⁺(maxi-K⁺) channelopening. ANG II-stimulated depolarized myocytes presented sustainedactivation of maxi-K⁺ channels,characterized by reduction from 415 to 12 ms of the closed timeconstant. ANG II desensitization was prevented by 100 nM iberiotoxin,being reversible within 30 min. Depolarization by KCl, higher than 4 mM, impaired desensitization, suggesting that the membrane potentialmust attain a threshold to counteract the repolarization induced bymaxi-K⁺ channel opening. Once thisvalue is attained, there is no time dependency because thedesensitization process was shut off by addition of KCl along the timecourse of the tonic response. In contrast, the sustained ACh toniccomponent was not altered by these maneuvers. We conclude thatdesensitization of the ANG II tonic component is foremost due to theopening of maxi-K⁺ channels,leading to membrane repolarization, thus closing the voltage-dependentCa²⁺ channels responsible for theCa²⁺ influx that sustains thetonic component in this muscle.

     

Ca2+ dependence and pharmacology of large-conductance K+ channels in nonlabor and labor human uterine myocytes

Two populations,Ca²⁺-dependent(BK_Ca) andCa²⁺-independentK⁺ (BK) channels of largeconductance were identified in inside-out patches of nonlabor and laborfreshly dispersed human pregnant myometrial cells, respectively.Cell-attached recordings from nonlabor myometrial cells frequentlydisplayed BK_Ca channel openings characterized by a relatively low open-state probability, whereas similar recordings from labor tissue displayed either no channel openings or consistently high levels of channel activity that oftenexhibited clear, oscillatory activity. In inside-out patch recordings,Ba²⁺ (2-10 mM),4-aminopyridine (0.1-1 mM), andShaker B inactivating peptide("ball peptide") blocked theBK_Ca channel but were much lesseffective on BK channels. Application of tetraethylammonium toinside-out membrane patches reduced unitary current amplitude ofBK_Ca and BK channels, withdissociation constants of 46 mM and 53 µM, respectively.Tetraethylammonium applied to outside-out patches decreased the unitaryconductance of BK_Ca and BKchannels, with dissociation constants of 423 and 395 µM,respectively. These results demonstrate that the properties of humanmyometrial large-conductance K⁺channels in myocytes isolated from laboring patients are significantly different from those isolated from nonlaboring patients.

     

Incubation of OKP cells in low-K+ media increases NHE3 activity after early decrease in intracellular pH

Chronichypokalemia increases the activity of proximal tubule apical membraneNa⁺/H⁺antiporter NHE3. The present study examined the effect ofthe incubation of OKP cells (an opossum kidney, clone P cell line) incontrol medium {K⁺ concn([K⁺]) = 5.4 mM} or low-K⁺ medium([K⁺] = 2.7 mM) onNHE3. The activity of an ethylisopropyl amiloride-resistant Na⁺/H⁺antiporter, whose characteristics were consistent with those ofNHE3, was increased inlow-K⁺ cells beginning at 8 h.NHE3 mRNA and NHE3 protein abundance were increased 2.2-fold and 62%,respectively, at 24 h but not at 8 h. After incubation inlow-K⁺ medium, intracellular pH(pH_i) decreased by 0.27 pH units(maximum at 27 min) and then recovered to the control level.Intracellular acidosis induced by 5 mM sodium propionate increasedNa⁺/H⁺antiporter activity at 8 and 24 h. Herbimycin A, a tyrosine kinase inhibitor, blocked low-K⁺- andsodium propionate-induced activation of theNa⁺/H⁺antiporter at 8 and 24 h. Our results demonstrate thatlow-K⁺ medium causes an earlydecrease in pH_i, which leads to anincrease in NHE3 activity via a tyrosine kinase pathway.     

Voltage-gated Ca2+ currents in rat gastric enterochromaffin-like cells

Enterochromaffin-like (ECL) cells are histamine-containingendocrine cells in the gastric mucosa that maintain a negative membranepotential of about 50 mV, largely due to voltage-gated K⁺ currents [D. F. Loo, G. Sachs, and C. Prinz. Am. J. Physiol. 270 (Gastrointest Liver Physiol. 33):G739-G745, 1996]. The current study investigated thepresence of voltage-gated Ca²⁺channels in single ECL cells. ECL cells were isolated from rat fundicmucosa by elutriation, density gradient centrifugation, and primaryculture to a purity >90%. Voltage-gatedCa²⁺ currents were measured insingle ECL cells using the whole cell configuration of the patch-clamptechnique. Depolarization-activated currents were recorded in thepresence of Na⁺ orK⁺ blocking solutions and additionof 20 mM extracellular Ca²⁺. ECLcells showed inward currents in response to voltage steps that wereactivated at a test potential of around 20 mV with maximalinward currents observed at +20 mV and 20 mM extracellular Ca²⁺. The inactivation rate of thecurrent decreased with increasingly negative holding potentials and wastotally abolished at a holding potential of 30 mV. Addition ofextracellular 20 mM Ba²⁺ insteadof 20 mM Ca²⁺ increased thedepolarization-induced current and decreased the inactivation rate. Theinward current was fully inhibited by the specific L-typeCa²⁺ channel inhibitor verapamil(0.2 mM) and was augmented by the L-typeCa²⁺ channel activator BAY K 8644 (0.07 mM). We conclude that depolarization activateshigh-voltage-activated Ca²⁺channels in ECL cells. Activation characteristics,Ba²⁺ effects, and pharmacologicalresults imply the presence of L-type Ca²⁺ channels, whereasinactivation kinetics suggest the presence of additional N-typechannels in rat gastric ECL cells.

     

Dependence of Na+-K+ pump current-voltage relationship on intracellular Na+, K+, and Cs+ in rabbit cardiac myocytes

To examine effects of cytosolicNa⁺, K⁺, and Cs⁺ on the voltagedependence of the Na⁺-K⁺ pump, we measuredNa⁺-K⁺ pump current (I_p)of ventricular myocytes voltage-clamped at potentials(V_m) from 100 to +60 mV. Superfusates weredesigned to eliminate voltage dependence at extracellular pump sites.The cytosolic compartment of myocytes was perfused with patch pipette solutions with a Na⁺ concentration ([Na]_pip)of 80 mM and a K⁺ concentration from 0 to 80 mM or withsolutions containing Na⁺ in concentrations from 0.1 to 100 mM and K⁺ in a concentration of either 0 or 80 mM. When[Na]_pip was 80 mM, K⁺ in pipette solutionshad a voltage-dependent inhibitory effect on I_pand induced a negative slope of theI_p-V_m relationship. Cs⁺ in pipette solutions had an effect onI_p qualitatively similar to that ofK⁺. Increases in I_p with increasesin [Na]_pip were voltage dependent. The dielectriccoefficient derived from[Na]_pip-I_p relationships at thedifferent test potentials was 0.15 when pipette solutions included 80 mM K⁺ and 0.06 when pipette solutions were K⁺ free.

     

IK channels are involved in the regulatory volume decrease in human epithelial cells

Parallel activation ofCa²⁺-dependent K⁺ channels and volume-sensitiveCl channels is known to be responsible for KCl effluxduring regulatory volume decrease (RVD) in human epithelial Intestine407 cells. The present study was performed to identify theK⁺ channel type. RT-PCR demonstrated mRNA expression ofCa²⁺-activated, intermediate conductance K⁺(IK), but not small conductance K⁺ (SK1) or largeconductance K⁺ (BK) channels in this cell line. Whole cellrecordings showed that ionomycin or hypotonic stress activated inwardlyrectifying K⁺ currents that were reversibly blocked by IKchannel blockers [clotrimazole (CLT) and charybdotoxin] but not by SKand BK channel blockers (apamin and iberiotoxin). Inside-out recordingsrevealed the existence of CLT-sensitive single K⁺-channelactivity, which exhibited an intermediate unitary conductance (30 pS at100 mV). The channel was activated by cytosolic Ca²⁺ ininside-out patches and by a hypotonic challenge in cell-attached patches. The RVD was suppressed by CLT, but not by apamin oriberiotoxin. Thus we conclude that the IK channel is involved in theRVD process in these human epithelial cells.