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PubMed1993

Propionate activates multiple ion transport mechanisms in the HT29-18-C1 human colon cell line.

Short-chain fatty acids (SCFAs) are the major solutes and the major anions in the colonic lumen. We studied the response of suspended HT29-18-C1 cells (an epithelial cell line derived from a human colon carcinoma) to SCFA exposure. Cellular response was evaluated by measurement of cell volume (Coulter counter), intracellular pH [pHi; measured fluorometrically with 2',7'-bis(2-carboxyethyl)-5-(6)-carboxyfluorescein (BCECF)], and intracellular Na+, K+, and Cl- content (flame photometry and chloride titrator). Exposure to 130 mM propionate in isosmotic medium causes a rapid decrease in pHi and activates pHi recovery via amiloride-sensitive Na-H exchange. In the presence of propionate, Na-H exchange also causes cell swelling to a peak volume 11% above control cells and causes a 2.8-fold increase in intracellular Na+ content. After peak swelling, a regulatory-volume decrease (RVD) significantly reduced volume and intracellular Na+ returned to baseline. Other SCFAs (acetate, butyrate, and valerate) also elicit swelling and RVD. Activation of the Na(+)-K(+)-adenosinetriphosphatase (ATPase) is required to return Na+ to normal levels and to indirectly provide ion gradients required for propionate-induced RVD, but Na(+)-K(+)-ATPase activity does not directly mediate RVD. When 1 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) is added in the presence of propionate, RVD was inhibited and cell Na+ content increased. Cl- depletion inhibited propionate-induced RVD and diminished the effect of SITS.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed2002

Pressure-dependent myogenic constriction of cerebral arteries occurs independently of voltage-dependent activation.

Pressure-induced decreases in arterial diameter are accompanied by membrane depolarization and Ca(2+) entry via voltage-gated Ca(2+) channels. Recent evidence also suggests the involvement of Ca(2+) sensitization of the contractile proteins. Both PKC and Rho kinase are candidate second messengers for the mediation of the sensitization process. We investigated the signaling pathways of pressure-induced decreases in rat cerebral artery diameter in vessels that were depolarized with a 60 mM potassium-physiological salt solution (KPSS). Arteries were mounted on a pressure myograph, and pressure-induced constrictions were recorded. In some experiments simultaneous changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) were recorded by using fura 2 fluorescence photometry. Pressure increases induced constriction with significant changes in [Ca(2+)](i) at high pressures (60-100 mmHg). The ratio of the change in diameter to change in [Ca(2+)](i) was greater for pressure-induced constriction compared with constriction produced by depolarization with 60 mM KPSS, suggesting that in addition to increases in [Ca(2+)](i), enhanced myofilament Ca(2+) sensitivity occurs during pressure-induced decreases in arterial diameter. Depolarizing the membrane with 60 mM KPSS increased [Ca(2+)](i) via a Ca(2+) influx pathway insensitive to PKC inhibition. Cerebral arteries were able to maintain their diameters in the continued presence of 60 mM KPSS. Pressure-induced constriction under these conditions was not associated with further increases in Ca(2+) but was abolished by selective inhibitors of PLC, PKC, and Rho kinase. We report for the first time that in rat cerebral arteries, pressure-induced decreases in arterial diameter are not only due to increases in voltage-gated Ca(2+) influx but also to accompanying increases in myofilament sensitivity to Ca(2+) mediated by PKC/Rho kinase activation.

Actin Cytoskeleton↗
PubMed2003

Evidence of a role for TRPC channels in VEGF-mediated increased vascular permeability in vivo.

Vascular endothelial growth factor (VEGF) increases vascular permeability by stimulating endothelial Ca(2+) influx. Here we provide evidence that links VEGF-mediated increased permeability and endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) with diacylglycerol (DAG)-mediated activation of the transient receptor potential channels (TRPCs). We used the Landis-Michel technique to measure changes in hydraulic conductivity (L(p)) and fluorescence photometry to quantify changes in endothelial [Ca(2+)](i) in individually perfused Rana mesenteric microvessels in vivo and transfected nonendothelial cells in vitro. The membrane-permeant DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG, 100 microM), which is known to increase Ca(2+) influx through TRPCs, transiently increased L(p) 3.8 +/- 1.2-fold (from 1.6 +/- 0.8 to 9.8 +/- 2.7 x 10(-7) cm.s(-1).cmH(2)O(-1); P < 0.0001; n = 18). Protein kinase C inhibition by bisindolylmaleimide (1 microM) did not affect the OAG-induced increases in L(p). OAG also significantly increased microvascular endothelial [Ca(2+)](i) in vivo (n = 13; P < 0.0001), which again was not sensitive to protein kinase C inhibition. VEGF induced a transient increase in endothelial [Ca(2+)](i) in human embryonic kidney cells (HEK-293) that were cotransfected with VEGF receptor 2 and TRPC-6 but not with control, VEGF receptor 2, or TRPC-6 expression vector alone (P < 0.01; n = 9). Flufenamic acid, which has been shown to enhance activity of TRPC-6 but inhibit TRPC-3 and -7, enhanced the VEGF-mediated increase in L(p) in approximately half of the vessels tested but inhibited the response in the other half of the vessels. These data provide evidence consistent with the hypothesis that VEGF increases vascular permeability via DAG-mediated Ca(2+) entry through TRPCs. Although the exact identities of the TRPCs remain to be confirmed, TRPC-6 appears to be a likely candidate in approximately half of the vessels.

Animals↗
PubMed1998

ACh- and caffeine-induced Ca2+ mobilization and current activation in rabbit arterial endothelial cells.

Fura 2 microfluorometry and perforated-patch whole cell recording were carried out simultaneously to investigate the relationship between intracellular free Ca2+ concentration ([Ca2+]i) and membrane current activation in response to ACh and caffeine in freshly dissociated arterial endothelial cells. ACh and caffeine evoked transient increases in [Ca2+]i. The initial increase in [Ca2+]i was accompanied by a transient outward current, which caused membrane hyperpolarization. The amplitudes of the [Ca2+]i transient and outward current were dependent on caffeine concentration (EC50 approximately 1 mM). Cyclopiazonic acid raised resting [Ca2+]i levels by >/=50 nM and failed to completely block caffeine- or ACh-induced [Ca2+]i transients but slowed [Ca2+]i recovery fourfold. The reversal potential of caffeine-induced currents was dependent on external K+ and Cl- concentrations. Caffeine-induced current amplitudes, but not [Ca2+]i responses, were attenuated by external tetraethylammonium, Zn2+, and La3+. A consistent temporal relationship between agonist-activated membrane current and [Ca2+]i increases was not observed, and, in some cases, time differences were greater than expected for simple diffusion of Ca2+ throughout the cell. These results suggest that Ca2+-dependent current activation monitors local [Ca2+]i changes adjacent to the plasmalemma, whereas single-cell photometry provides a measure of global changes in [Ca2+]i.

Acetylcholine↗
PubMed1999

Sarcoplasmic reticulum and endothelium independently regulate venous smooth muscle [Ca(2+)](i) and contraction.

In rings of rabbit facial vein (RFV), depletion of sarcoplasmic reticulum (SR) Ca(2+) by caffeine abolished the subsequent isometric contraction to 25 mM K(+) physiological salt solution (25K-PSS). However, the associated steady-state increase of smooth muscle intracellular free Ca(2+) concentration ([Ca(2+)](i)), measured using fura PE3 and cuvette photometry, was not altered. Treatment with the specific SR Ca(2+) pump inhibitor cyclopiazonic acid (30 microM) after caffeine-induced SR Ca(2+) depletion restored and greatly augmented the 25K-PSS-induced contraction. This suggests that SR Ca(2+) depletion leads to a dissociation of K(+)-induced [Ca(2+)](i) increase from contraction that was dependent on Ca(2+) pump-mediated SR Ca(2+) uptake. Endothelium removal augmented the 25K-PSS-induced [Ca(2+)](i) increase after caffeine-induced SR Ca(2+) depletion. However, this was associated with only a small and transient contraction. Exposure of endothelium-denuded RFV to cyclopiazonic acid after caffeine-induced SR Ca(2+) depletion further amplified the 25K-PSS-induced [Ca(2+)](i) increase, which was associated with a large and sustained contraction. However, the latter [Ca(2+)](i) increase was still higher than in endothelium-intact RFV. This suggests that the endothelium dampens the [Ca(2+)](i) rise associated with K(+)-induced Ca(2+) influx, but independently of Ca(2+) pump-mediated SR Ca(2+) uptake.

Animals↗
PubMed1975

Localization of thirst and antidiuretic osmoreceptors by intracranial injections in rats.

Eighty-six intracranial cannula placements in 51 rats were tested with unilateral, 2-mul injections of a 0.60-osmol/kg solution, sucrose dissolved in isotonic sodium chloride. To assess antidiuretic hormone (ADH) release, a water diuresis was induced and spontaneous urinations were collected and analyzed for sodium by flame photometry. On alternate test days the 0.60-osmol/kg solution was injected into sleeping rats, and latencies to drink and volume drunk were recorded. Injections at 42 placements elicited neither drinking nor adtidiuresis on two separate test days each; at 15, both antidiuresis and drinking on at least two of three tests each; at 19, drinking but not antidiuresis; at 10, antidiuresis but not drinking. Positive drinking and ADH placements were not distinctly separated. They clustered in the bed nucleus of the stria terminalis, the preoptic areas, and the anterior portions of the hypothalamus. Placements in the medial forebrain bundle and dorsal to the anterior hypothalamic area elicited thirst but not ADH release for the most part. Placements nearest the supraoptic nucleus were weak or negative for ADH release. Central nervous system osmo-receptors exist and seem not to be the neurosecretorycells. Thirst osmoreceptors and antidiuretic osmoreceptors seem to be contiguous, but distinct.

Angiotensin II↗
PubMed2003

Effects of chloride channel blockers on rat renal vascular responses to angiotensin II and norepinephrine.

The aim of the present study was to investigate the role of Ca2+-activated Cl- channels in the renal vasoconstriction elicited by angiotensin II (ANG II) and norepinephrine (NE). Renal blood flow (RBF) was measured in vivo using electromagnetic flowmetry. Ratiometric photometry of fura 2 fluorescence was used to estimate intracellular free Ca2+ concentration ([Ca2+]i) in isolated preglomerular vessels from rat kidneys. Renal arterial injection of ANG II (2-4 ng) and NE (20-40 ng) produced a transient decrease in RBF. Administration of ANG II (10-7 M) and NE (5 x 10-6 M) to the isolated preglomerular vessels caused a prompt increase in [Ca2+]i. Renal preinfusion of DIDS (0.6 and 1.25 micromol/min) attenuated the ANG II-induced vasoconstriction to approximately 35% of the control response, whereas the effects of NE were unaltered. Niflumic acid (0.14 and 0.28 micromol/min) and 2-[(2-cyclopentenyl-6,7-dichloro-2,3-dihydro-2-methyl-1-oxo-1H-inden-5-yl)oxy]acetic acid (IAA-94; 0.045 and 0.09 micromol/min) did not affect the vasoconstrictive responses of these compounds. Pretreatment with niflumic acid (50 microM) or IAA-94 (30 microM) for 2 min decreased baseline [Ca2+]i but did not change the magnitude of the [Ca2+]i response to ANG II and NE in the isolated vessels. The present results do not support the hypothesis that Ca2+-activated Cl- channels play a crucial role in the hemodynamic effects of ANG II and NE in rat renal vasculature.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2004

Effect of tyrosine kinase blockade on norepinephrine-induced cytosolic calcium response in rat afferent arterioles.

We used genistein (Gen) and tyrphostin 23 (Tyr-23) to evaluate the importance of tyrosine phosphorylation in norepinephrine (NE)-induced changes in intracellular free calcium concentration ([Ca(2+)](i)) in rat afferent arterioles. [Ca(2+)](i) was measured in microdissected arterioles using ratiometric photometry of fura 2 fluorescence. The control [Ca(2+)](i) response to NE (1 microM) consisted of a rapid initial peak followed by a plateau phase sustained above baseline. Pretreatment with the tyrosine kinase inhibitor Tyr-23 (50 microM, 10 min) caused a slow 40% increase in baseline [Ca(2+)](i). Tyr-23 attenuated peak and plateau responses to NE, both by approximately 70%. In the absence of extracellular Ca(2+) (0 Ca), Tyr-23 reduced the immediate [Ca(2+)](i) response to NE by approximately 60%, indicative of mobilization of internal stores, and abolished the plateau phase. In other arterioles, the [Ca(2+)](i) response to depolarization induced by KCl (50 mM) was not attenuated by Tyr-23, indicating no direct effect on L-type Ca(+) channels activated by depolarization. The Ca(2+) channel blocker nifedipine (1 microM) inhibited the NE response by approximately 50%; the effects of nifedipine and Tyr-23 were not additive. Nifedipine had no inhibitory effect after Tyr-23 pretreatment, indicating Tyr-23 inhibition of Ca(2+) entry. Another tyrosine kinase inhibitor, Gen (5 and 50 microM), did not affect baseline [Ca(2+)](i). High-dose Gen inhibited the peak and plateau response to NE by 87 and 75%, respectively; low-dose Gen attenuated both responses by approximately 20%. In 0 Ca, Gen (50 microM) abolished the immediate [Ca(2+)](i) mobilization response. Combined nifedipine and Gen (50 microM) inhibited the rapid NE response by approximately 90% in the presence of extracellular Ca(2+). Gen (50 microM) also inhibited by 60% the [Ca(2+)](i) response to 50 mM KCl, indicating a direct interaction with voltage-sensitive, L-type Ca(2+) entry channels. These results indicate that tyrosine phosphorylation is an important link in the chain of events leading to alpha-adrenoceptor-induced Ca(2+) recruitment (both entry and release) in afferent arteriolar smooth muscle cells. Furthermore, different blockers of tyrosine kinase appear to have different modes of action in renal microvessels.

Animals↗
PubMed1982

Na+ transport properties of the peritubular membrane of cortical collecting tubule.

The effects of varying endogenous aldosterone levels on the passive and active properties of the peritubular membrane were studied. Rabbits that were fed either a low Na+ (normal K+) diet or a high Na+, low K+ diet increased or decreased plasma aldosterone, respectively. Tubules were dissected, filled with oil, and incubated in 0 K+ medium to increase intracellular sodium. Cellular sodium and potassium content was measured by helium-glow photometry. The degree to which cells accumulate sodium and lose potassium is a function not only of time of exposure but also of diet. Tubules from animals on a low Na+ diet are about 6 times more permeable to sodium than those from animals fed a high Na+ diet. When tubules were loaded with sodium and returned to a normal (5 mM K+) bathing medium, net sodium efflux and potassium influx occurred. The rate of sodium efflux by cortical collecting tubules dissected from animals on the low Na+ diet was 2.3 times greater than the efflux rate of tubules from animals on the high Na+ diet. These data suggest that high levels of endogenous aldosterone enhance sodium transport measured in vitro across the peritubular membrane of cortical collecting tubule.

Animals↗
PubMed1984

Direct measurement of potassium leak from single 3 M KCl microelectrodes.

Potassium loss was measured from individual microelectrodes filled with 3 M KCl. The potassium content of a distilled-water droplet into which a microelectrode was inserted for a known time interval was analyzed by helium-glow photometry. The rate of potassium loss from single microelectrodes (18-97 M omega) was within the detectable limits of this method. The rate of potassium loss appeared to decrease with increasing input resistance (determined in 0.16 M NaCl) for electrodes having a low resistance (18-50 M omega). Higher resistance electrodes (greater than 50 M omega) appeared to achieve a uniform rate of potassium loss of 3.5 fmol X s-1. Estimates of the rate of potassium loss calculated from the rate of swelling of impaled cells were identical to those measured directly on the same group of electrodes. We conclude the electrolyte loss not only occurs into a distilled-water drop but also during intracellular recording. An alternative method for fabrication of microelectrodes was tested. Of these electrodes, the ones having an input resistance greater than 50 M omega lost potassium at rates below 1 fmol X s-1.

Animals↗
PubMed1987

K transport in upper portion of descending limbs of long-loop nephron from hamster.

By use of the in vitro microperfusion technique, we investigated potassium transport in the upper portion of the descending limb of the long-loop nephron isolated from hamster kidney. The net potassium flux determined by ultramicro-flame photometry was -0.63 +/- 1.84 pmol X mm-1 X min-1, a value that was not significantly different from zero. The salt permeability for KCl was calculated from the amount of potassium entering the tubular lumen when the concentration of potassium in the bath was increased by approximately 5 mM. The value was 38.9 +/- 1.9 X 10(-5) cm X s-1. The bidirectional fluxes of 86Rb were measured as indices of potassium fluxes. Flux coefficients from lumen-to-bath and bath-to-lumen were 51.2 +/- 9.2 and 48.8 +/- 13.5 X 10(-5) cm X s-1, respectively. These values were not significantly different, confirming that there was no net flux for 86Rb. In another series of five experiments, the lumen-to-bath 86Rb flux coefficients were 69.4 +/- 13.2 X 10(-5) cm X s-1 in the absence of unlabeled Rb, and 70.2 +/- 13.9 X 10(-5) cm X s-1 in the presence of 5 mM unlabeled Rb. The lumen-to-bath 42K flux coefficient measured in the same series of animals was 85.3 +/- 10.5 X 10(-5) cm X s-1 (n = 10), a value that is slightly higher than, but not significantly different from, that of 86Rb. These data show that active potassium transport may exist in this segment and that the passive permeability for potassium is very high.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

NMR monitoring of intracellular sodium in dog and rabbit kidney-tubules.

23Na-nuclear magnetic resonance (NMR) was used to monitor intra- and extracellular sodium in suspensions of dog cortical tubules, rabbit cortical tubules, and dog thick ascending limbs. The NMR visibility of the intracellular sodium was determined by comparing the NMR and flame photometry results and by redistributing the sodium ions between the intra- and extracellular compartments using the ionophore nystatin (influx) or sodium substitution for choline in the extracellular fluid (efflux). The intracellular sodium visibility was approximately 30% for the total sodium and 58% for the transportable sodium. Addition of sodium to sodium-depleted homogenates of dog renal cortex also showed a loss of visibility. The values of the relaxation times T1 and T2 were determined but could not be correlated with the visibility measurements. The intracellular sodium concentration in dog cortical tubules incubated in optimal biochemical conditions was estimated at 51 mM and was dependent on the extracellular sodium concentration.

Animals↗
PubMed1987

The collecting tubule of Amphiuma. II. Effects of potassium adaptation.

Electrophysiological and transport properties were studied in isolated and perfused Amphiuma collecting tubules from two groups of animals, one exposed to a high sodium (NA), the other to a high potassium (KA) environment (both conditions known to modulate blood aldosterone levels). The transepithelial lumen-negative potential was significantly larger (-38 +/- 5 mV) in tubules from KA animals than from NA animals (-15 +/- 3 mV). In addition, we observed an increase in the apical amiloride-sensitive sodium conductance and stimulation of the transepithelial sodium current. Although no measurable potassium conductance was found in the apical cell membrane in either group, a potassium selectivity of the paracellular transport pathway was observed in the KA animals. Net potassium secretion was demonstrated in KA tubules (helium-glow photometry and [3H]inulin analysis). Potassium secretion was abolished by luminal amiloride but imposition of a bath-to-lumen potassium gradient induced potassium secretion. We conclude that in contrast to the mammalian cortical collecting tubule in which potassium secretion is largely transcellular, potassium secretion in the Amphiuma collecting tubule is by diffusion through the paracellular pathway.

Adaptation, Physiological↗
PubMed1988

Potassium adaptation: 39K-NMR evidence for intracellular compartmentalization of K+.

To investigate the effects of K+ uptake on the intracellular environment, both 39K-nuclear magnetic resonance (NMR) and K+-selective electrodes were used to measure K+ activity with acute K+ loading in control and K+-adapted rats. These results were then compared with tissue K+, measured by flame photometry. There was a lower NMR K+ visibility (ratio of NMR signal to tissue content) in muscle and liver in K+-adapted rats, compared with controls before and after an acute K+ load. This lower K+ visibility in K+-adapted rats was confirmed in liver homogenate with the K+-specific electrode. In liver homogenates from control and K+-adapted rats, addition of RbCl (300 mumol/g) increased the NMR K+ signal more in K+-adapted rats (19 +/- 1.1 mumol/g) than controls (11 +/- 1.0 mumol/g, P less than 0.01). This is consistent with the displacement of K+, by Rb+, from NMR-undetected sites. These results suggest that some 10-15% of intracellular K+ may be within a compartment not detectable by NMR or electrodes and that chronic K+ loading leads to an increased capacity of this compartment.

Adaptation, Physiological↗
PubMed1993

Oxytocin affects apical sodium conductance in rabbit cortical collecting duct.

The physiological role of oxytocin (OT) in the kidney is still unclear, although autoradiographic data have shown the existence of OT receptors in the rat kidney. We examined the effect of OT in the microperfused rabbit cortical collecting duct (CCD) by using conventional cable analysis and microscope photometry. On addition of 10(-9) M OT to the bath, the lumen-negative transepithelial voltage (VT) transiently increased and the transepithelial resistance (RT) and the fractional resistance of the apical membrane (FRA) (1st phase) both decreased. After this initial change, the lumen-negative VT gradually decreased below its baseline level and RT and FRA (second phase) both increased. These electrical changes were dose dependent and were prevented by the addition of 10(-5) M amiloride to the lumen. Although responses to OT were not prevented by 10(-9) M arginine vasopressin (AVP) or 10(-6) M of a V1-receptor antagonist (OPC-21268) or V2-receptor antagonist (OPC-31260), they were inhibited by the addition of the specific OT antagonist des-Gly-NH2-[d(CH2)3,Tyr(Me),Thr]OVT. Additional studies of intracellular free calcium ([Ca2+]i) revealed that 10(-8)-10(-6) M OT caused an increase in [Ca2+]i in CCD in a dose-dependent manner. Also, pretreatment with 2 x 10(-8) M bis-(aminophenoxy)ethane-tetraacetic acid-acetoxymethyl ester, an intracellular Ca2+ chelator, abolished the electrical and [Ca2+]i responses to OT. Pretreatment with 5 x 10(-4) M 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP) partially prevented the electrical responses to OT, thus reducing the decrease in lumen-negative VT below its basal level and the increase in RT after the 1st phase. These data show that OT affects the apical Na+ conductance of collecting duct cells through OT receptors distinct from the AVP receptors and that the effect of OT may, at least in part, be brought about by a mechanism(s) dependent on the increase in [Ca2+]i and cAMP production.

Animals↗
PubMed1994

Postnatal maturation of potassium transport in rabbit cortical collecting duct.

Clearance studies in newborns demonstrate low rates of urinary excretion of potassium, suggesting that the neonatal kidney contributes to the conservation of potassium necessary for growth. Because the cortical collecting duct (CCD) is a primary site for potassium secretion in the adult, we sought to examine the transport capacity of this segment for potassium during postnatal maturation. CCDs isolated from rabbits of various ages (5-6 animals/age group) were microperfused in vitro with solutions simulating plasma. The concentrations of potassium in samples of collected fluid, measured by helium glow photometry, were used to calculate net transport. At a flow rate of approximately 1.6 nl.min-1 x mm-1 net potassium secretion was absent at birth, first became evident at 4 wk of age (-11.08 +/- 2.39 pmol.min-1 x mm-1), and increased sharply thereafter to reach mature rates (-23.08 +/- 3.47 pmol.min-1 x mm-1; P < 0.05) by 6 wk of age. To determine whether low distal tubular flow rates limit net potassium secretion in the neonate, we perfused CCDs at two or more flow rates in the 0.5-5 nl.min-1 x mm-1 range. In CCDs taken from animals > or = 6 wk of age, potassium secretion showed a significant linear correlation with flow rate (y = -10.0x - 7.45; r = 0.87; n = 12).(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗
PubMed1994

Axial heterogeneity of potassium transport across hamster thick ascending limb of Henle's loop.

Functional significance of morphological heterogeneities along the thick ascending limb of Henle's loop of hamsters was explored by the in vitro microperfusion technique with special reference to K+ transport. The transmission electron microscopic study confirmed that there are two types of cells, with smooth surface (S-cell) and rough surface (R-cell), respectively, and that the former is abundant in the medullary thick ascending limb (MTAL), whereas the latter is in the cortical portion (CTAL). The electrophysiological study revealed that in both segments there are two cell populations, one having high basolateral and low apical membrane K+ conductances (HBC) and the other having low basolateral and high apical K+ conductances (LBC). Random cell puncture revealed that the ratios of HBC/LBC were 24/7 (77%/23%) in the MTAL and 7/22 (24%/76%) in the CTAL, suggesting that HBC corresponds to S-cell, whereas LBC corresponds to R-cell. Net K+ transport was determined in two segments by measuring K+ concentration in the collected and perfused fluid by ultramicroflame photometry. In all six tubules of MTAL, net K+ flux had a direction to reabsorption with a mean of 4.87 +/- 0.46 pmol.min-1.mm-1. In marked contrast, in all six tubules of CTAL, we observed K+ secretion with a mean of -3.81 +/- 0.49 pmol.min-1.mm-1. The transmural voltage was positive in both segments and was significantly higher in the CTAL (7.8 +/- 0.5 mV) than in the MTAL (2.5 +/- 0.2 mV). From these observations, we conclude that the S-cell corresponding to the HBC cell reabsorbs K+, whereas the R-cell corresponding to the LBC cell secrets K+.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

Angiotensin II-receptor stimulation of cytosolic calcium concentration in cultured renal resistance arterioles.

This study provides an initial characterization of basic morphological properties of cultures of vascular smooth muscle cells (VSMC) from rat preglomerular resistance vessels and of the functional coupling of angiotensin II (ANG II) receptors to cytosolic free calcium concentration ([Ca2+]i (fura 2 fluorescence photometry). Renal VSMC were isolated from interlobular arteries and afferent arterioles (< 50 microns) using an iron oxide sieving method and compared with rat aortic VSMC cultured under similar conditions. Quiescent monolayers maintained uniform morphology and [Ca2+]i signaling profile between passages 3 and 10. Arteriolar and aortic VSMC were spindle shaped and expressed smooth muscle-specific alpha-actin and myosin heavy chains SM-1 and SM-2. ANG II caused a rapid increase in [Ca2+]i, followed by a sustained plateau phase at 50-60% of the peak value. The initial maximum [Ca2+]i responses were dose dependent and of similar magnitude in renal arteriolar and aortic VSMC. ANG II (10(-7) M) increased [Ca2+]i from 50 to 240 nM in arteriolar and from 57 to 201 nM in aortic VSMC (P < 0.001 for both). Inhibition of ANG II effects on [Ca2+]i revealed significant signaling through distinct AT-receptor subtypes (losartan and PD-123319 sensitive) in renal arteriolar VSMC. In contrast, only losartan was effective in aortic VSMC. The AT2-receptor ligand CGP-42112 had no effect in either vessel type. Our results demonstrate that cultured arteriolar VSMC have anatomical similarities to aortic VSMC and functional differences in AT-receptor signaling in response to ANG II. This novel preparation should provide a useful approach with which to investigate cellular mechanisms concerning receptor coupling to signaling pathways involved in vascular reactivity of arteriolar VSMC in the microcirculation in general and the kidney in particular.

Animals↗