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J L Garvin

Publications and source records attributed to J L Garvin.

At least 55 records · Page 3Linked to original sources
PubMed1992

ANF and bradykinin synergistically inhibit transport in M-1 cortical collecting duct cell line.

Previous data suggest that atrial natriuretic factor (ANF) and bradykinin (BK) interact to increase Na+ and water excretion. We propose that this interaction is due to a synergistic action that inhibits Na+ absorption in the distal nephron. We examined the effects of BK and ANF on transport by monolayers of a cortical collecting duct cell line, M-1. BK (10(-8) M) had no effect on short-circuit current (Isc). Similarly, ANF (10(-8) M) did not inhibit Isc. In contrast, Isc decreased by 18% (from 57 +/- 8 to 46 +/- 6 microA/cm2) when BK and ANF were added simultaneously at this concentration (P less than 0.05). Because guanosine 3',5'-cyclic monophosphate (cGMP) and protein kinase C are implicated in the second messenger cascades of ANF and BK, we investigated their potential roles in mediating this interaction. Dibutyryl-cGMP (10(-4) M) inhibited Isc from 33 +/- 4 to 22 +/- 3 microA/cm2 (P less than 0.05) in the presence of BK but not in its absence. Staurosporine and calphostin C, inhibitors of protein kinase C, completely blocked the decrease in Isc caused by simultaneous addition of ANF and BK. cAMP levels in M-1 cells were not affected by either ANF alone or BK alone; however, when cultures were treated with both hormones, cAMP decreased from 856 +/- 56 to 332 +/- 26 fmol/10(6) cells (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

ANF inhibits norepinephrine-stimulated fluid absorption in rat proximal straight tubules.

We have previously shown that atrial natriuretic factor (ANF) only inhibits fluid absorption in the proximal straight tubule after the tissue has been exposed to angiotensin (ANG) II. In this paper, the interaction between ANF and norepinephrine (NE) in the proximal straight tubule was investigated. ANF alone (10(-9) M) added to the bath had no significant effect on fluid absorption. In contrast, when tubules were first treated with 10(-7) M NE, 10(-9) M ANF reduced fluid absorption from 0.63 +/- 0.09 to 0.41 +/- 0.04 nl.mm-1.min-1. NE alone stimulated fluid absorption by 33%. Dibutyryl-guanosine 3',5'-cyclic monophosphate (cGMP; 50 microM) mimicked the effects of ANF, reducing fluid absorption from 0.73 +/- 0.06 to 0.35 +/- 0.07 nl.mm-1.min-1 in NE-treated tubules. However, it had no effect on fluid absorption in the absence of NE. These studies suggest that 1) ANF can regulate fluid absorption in the proximal nephron; 2) this inhibition occurs only after transport has been stimulated by agents such as NE (or ANG II); 3) cGMP is part of the second messenger system of ANF in the rat proximal straight tubule; and 4) the primary interaction between ANF and NE (or ANG II) occurs in the second messenger cascades subsequent to steps that affect levels of cGMP.

Absorption↗
PubMed1992

Endothelium-derived relaxing factor inhibits transport and increases cGMP content in cultured mouse cortical collecting duct cells.

Stimulation of the release of endothelium-derived relaxing factor (EDRF) in the kidney has been shown to result in natriuresis without affecting glomerular filtration rate. This may be due to EDRF directly regulating solute transport in the cortical collecting duct (CCD). To test this hypothesis, we measured the effect of bradykinin (Bk) or acetylcholine (Ach) on short-circuit current (Isc; a measure of active transport) in a CCD cell line (M-1), in the presence or absence of cow pulmonary artery endothelial (CPAE) cells. 10(-9) M Bk or 10(-7) M Ach had no effect on M-1 Isc in which CPAE cells were absent. The addition of CPAE cells to M-1 cells also did not affect M-1 Isc. On the other hand, when 10(-9) M Bk or 10(-7) M Ach were added to M-1 cells in the presence of CPAE cells, Isc decreased from 43 +/- 4.5 to 26 +/- 4 and 64 +/- 9 to 33 +/- 4 microA/cm2, respectively (P less than 0.001). Nitroarginine (N-Arg, 10(-4) M), a competitive inhibitor of EDRF production, blocked the inhibition in M-1 Isc due to both agonists. Since cGMP is the second messenger of EDRF in vascular smooth muscle, we measured the effects of Bk on cGMP production in M-1 cells in the presence and absence of CPAE cells. Bk increased cGMP content in M-1 cells in the presence of CPAE cells from 33 +/- 3.4 to 132 +/- 11.7 fmol/10(6) M-1 cells (P less than 0.001). When cultures of M-1 and CPAE cells were treated with N-Arg and challenged with Bk, Bk's effect on cGMP was partially blocked (61.4 +/- 12 fmol/10(-6) M-1 cells; NS). These data suggest that EDRF inhibits transport and increases cGMP content in M-1 cells.

Acetylcholine↗
PubMed1991

Angiotensin stimulates bicarbonate transport and Na+/K+ ATPase in rat proximal straight tubules.

The effect of angiotensin on HCO3- absorption, fluid absorption, and Na+/K+ ATPase activity in isolated rat proximal straight tubules was investigated. During the control period, tubules absorbed fluid at 0.66 +/- 0.12 nL/mm.min and bicarbonate at 60.2 +/- 10.7 pmol/mm.min. After 10(-10) M angiotensin was added to the bath, tubules absorbed fluid at 0.93 +/- 0.19 nL/mm.min and bicarbonate at 77.4 +/- 15.2 pmol/mm.min, indicating stimulation of both parameters. Time controls showed no significant change in the rate of bicarbonate or fluid absorption. To determine whether this stimulation was due to an increase in the maximum rate of transport, tubules were perfused at greater than or equal to 20 nL/mm.min. During the control period, tubules absorbed bicarbonate at 82.5 +/- 13.0 pmol/mm.min. After 10(-10) M angiotensin was added to the bath, these same tubules absorbed bicarbonate at 75.9 +/- 11.9 pmol/mm.min. Thus, angiotensin did not alter the maximum rate of transport. Angiotensin also had no effect on bicarbonate permeability, which was 1.1 +/- 0.2 x 10(-4) cm/s before treatment and 1.3 +/- 0.3 x 10(-4) cm/s afterward. Finally, the effect of angiotensin on Na+/K+ ATPase activity was measured in paired experiments. Na+/K+ ATPase activity of control tubules was 36 +/- 6 pmol of ADP/mm.min; after angiotensin treatment, it was 47 +/- 6 pmol ADP/mm.min. From these data it was concluded that: (1) angiotensin stimulates bicarbonate absorption in the rat proximal straight tubule; (2) this stimulation is the result of a change in Km rather than of an increase in the maximum rate of transport or permeability; and (3) angiotensin directly stimulates Na+/K+ ATPase activity in the proximal nephron.

Absorption↗
PubMed1990

Glucose absorption by isolated perfused rat proximal straight tubules.

Glucose absorption was investigated in isolated perfused proximal straight tubules from rats by use of a newly developed ultramicrofluorometric assay. This assay takes advantage of the increase in fluorescence associated with the reduction of NAD to NADH while glucose is degraded to 6-phosphogluconate. When tubules were perfused at 6.70 +/- 0.42 nl.mm-1.min-1, the mean rate of glucose absorption was 11.0 +/- 1.0 pmol.mm-1.min-1, and the mean rate of fluid absorption was 0.61 +/- 0.06 nl.mm-1.min-1. Glucose transport is generally due to Na-glucose cotransport in the proximal nephron. In the rat proximal straight tubule, glucose absorption also appeared to be primarily due to Na-glucose cotransport, since 10(-4) M phlorizin inhibited absorption by 100%, as did inhibition of Na(+)-K(+)-ATPase by K removal. To determine the maximum rate of transport, tubules were perfused at rates greater than 20 nl.mm-1.min-1 with a solution containing 5.5 mM glucose. The maximum rate of glucose absorption was approximately 20 pmol.mm-1.min-1 under these conditions. The concentration of glucose that supports 50% of the maximum rate of absorption, Km, was 0.6 mM. When tubules were perfused at flow rates of less than or equal to 2 nl.mm-1.min-1, the luminal glucose concentration reached a limiting value of 0.47 mM with 5.5 mM glucose in the bath. The glucose permeability was 3.1 X 10(-6) cm/s.

Absorption↗
PubMed1990

Angiotensin stimulates glucose and fluid absorption by rat proximal straight tubules.

I investigated the effect of angiotensin on glucose and fluid absorption by isolated, perfused rat proximal straight tubules. During the control period, tubules absorbed fluid at 0.68 +/- 0.05 nL/mm.min and glucose at 12.5 +/- 1.3 pmol/mm.min. After 10(-10) M angiotensin was added to the bath, tubules absorbed fluid at 0.82 +/- 0.06 nL/mm.min and glucose at 16.0 +/- 2.4 pmol/mm.min--a stimulation of both parameters by 30%. Time controls showed no significant change in the rate of glucose or fluid absorption. This stimulation was due to an increase in the maximum rate of transport. The maximum rate of glucose absorption with 5.5 mM in bath and perfusate increased from 14.5 +/- 1.6 pmol/mm.min to 18.5 +/- 1.4 pmol/mm.min after 10(-10) M angiotensin was added to the bath. In contrast, the Km for glucose did not change, since a solution containing 0.55 mM glucose (approximately Km) supported 63 +/- 6% of the maximum rate during the control period and 60 +/- 5% of the maximum rate after angiotensin was added to the bath. Angiotensin also had no effect on glucose permeability, which was (3.7 +/- 0.4) x 10(-6) cm/s before treatment and (3.8 +/- 0.4) x 10(-6) cm/s after treatment. From these data, it is concluded that: (1) angiotensin stimulates glucose absorption in the rat proximal straight tubule; (2) this stimulation is the result of an increase in the maximum rate of transport rather than a change in Km or permeability; and (3) stimulation of Na/glucose absorption by angiotensin accounts for a significant portion of the increase in fluid absorption caused by angiotensin.

Absorption↗
PubMed1989

Inhibition of Jv by ANF in rat proximal straight tubules requires angiotensin.

The effects of atrial natriuretic factor (ANF) on fluid absorption (Jv) by isolated perfused proximal straight tubules of rats were investigated. ANF alone (10(-8) M) added to the bath had no significant effect on absorption. In contrast, when tubules were first treated with 1.6 X 10(-10) M angiotensin II, this same concentration of ANF lowered fluid absorption from 0.99 +/- 0.03 to 0.69 +/- 0.02 nl.mm-1.min-1. A lower dose of ANF, 2 X 10(-10) M, reduced fluid absorption in the presence of angiotensin II from 1.13 +/- 0.06 to 0.65 +/- 0.05 nl.mm-1.min-1, an inhibition of 40%. Since guanosine 3',5'-cyclic monophosphate (cGMP) is reportedly part of the second messenger system of ANF, the effects of dibutyryl-cGMP (DBcGMP) on fluid absorption were studied. This membrane-permeant form of cGMP mimicked the effects of ANF, reducing fluid absorption from 1.15 +/- 0.18 to 0.54 +/- 0.08 nl.mm-1.min-1. These studies suggested the following: 1) ANF can regulate fluid absorption in the proximal nephron; 2) this inhibition occurs only in the presence of angiotensin; and 3) cGMP is part of the second messenger system of ANF in the rat proximal straight tubule, as it is in other tissues.

Absorption↗
PubMed1988

Spatial map of salts and saccharides on dog tongue.

Front and rear regions of dog tongue were tested for spatial heterogeneity with respect to monovalent chloride salts (NaCl, KCl, and NH4Cl) and sweet tastants (sucrose, D-glucose, and L-glucose) by placing them in Ussing chambers where the open-circuit potential, Voc, and short-circuit current, Isc, were measured. The responses of Voc and Isc to 0.5 M NaCl were greater in the front of the tongue than in the rear, whereas the responses of Voc and Isc to 0.5 M sucrose were greater in the rear of the tongue than in the front. These results are similar to those of published neurophysiological measurements from the chorda tympani nerve and thalamus. These data suggest that the spatial heterogeneities seen in higher taste centers are present in the isolated lingual epithelium where primary taste transduction events occur.

Ammonium Chloride↗
PubMed1988

Active NH4+ absorption by the thick ascending limb.

Isolated perfused medullary thick ascending limbs from rabbits were studied to determine the mechanism of ammonium ion absorption. Under control conditions, thick ascending limbs spontaneously absorbed NH4+ and generated a lumen-positive potential. When these tubules were chemically voltage clamped to lumen-negative potentials by lowering the bath NaCl concentration, NH4+ absorption persisted. Thus NH4+ was absorbed against an electrochemical gradient. The active flux accounts for most of the net flux under control conditions, the remainder being due to passive paracellular NH4+ diffusion. The NH4+ permeability, measured in separate experiments, was high (1.50 +/- 0.25 x 10(-4) cm/s) compared with values in other segments. The NH3 permeability was relatively low (3.1 +/- 0.5 x 10(-3) cm/s). Luminal furosemide (10(-4) M) eliminated most of the active NH4+ flux, indicating that a major fraction of the active flux is dependent on apical entry of NH4+ via the Na+ -K+ -2Cl- cotransporter (presumably by substitution for K+). The remaining active flux was completely inhibited by 10(-4) M ouabain in the bath. Active chloride absorption was maintained when NH4+ entirely replaced K+ in bath and perfusate, indicating that NH4+ substitutes for K+ on the apical cotransporter and the basolateral Na+ -K+ -ATPase. Ammonium absorption provides an active "single effect" for countercurrent multiplication of NH4+ in the renal medulla.

Absorption↗
PubMed1988

Secretion of endolymph by semicircular canals of the shark.

The semicircular canals of the vestibular labyrinth of the dogfish shark, Squalus acanthias, may serve as a simple in vitro system for the study of the ionic transport mechanisms involved in endolymph formation. Electron microscopy showed that the epithelium was made up of at least three distinct cell types divided into separate regions running the length of the canals. Secretion of endolymph was studied in isolated canals by the split droplet method; when the lumens of the canals were filled with shark Ringer a potassium-rich fluid was secreted into the lumen at a rate of 0.34 microliter.cm-2.min-1. The K concentration of the secreted fluid averaged 112 mM; the calculated rate of K secretion was 2.3 mumol.cm-2.h-1, comparable to recent measurements in mammalian utricle. Fluid secretion was dependent on active transport and was inhibited by ouabain, bumetanide, or methazolamide in the external bathing solution. Fluid secretion was unaffected by the K channel blocker, Ba, in the luminal droplet or by the adenylate cyclase stimulator, forskolin, in the external bathing solution. For electrophysiological analysis, isolated canals were perfused in a chamber designed for voltage/current clamping; an axial wire was inserted into the canal lumen and constant-current pulses were passed to determine tissue resistance. When the luminal fluid was high-K Ringer, transepithelial potential difference was -1.1 mV (lumen negative) and resistance 37 omega.cm2. Dilution and bi-ionic potential measurements showed that the epithelium of the canals exhibited only slight cation selectivity. These results are consistent with a model for endolymph secretion involving cotransport secondary to the ion gradients created by the Na+-K+-ATPase.

Animals↗
PubMed1987

NH3 and NH4+ transport by rabbit renal proximal straight tubules.

UNLABELLED: Isolated perfused S2 proximal straight tubules from rabbits spontaneously secreted ammonia (-1.34 pmol X mm-1 X min-1) and absorbed bicarbonate (49.3 pmol X mm-1 X min-1) when perfusate and bath solutions contained 1 mM NH4Cl and 25 mM bicarbonate (pH 7.4). The NH3 concentration in the collected fluid was on average 40% lower than that of the bath as a consequence of a lower pH in the lumen. To test whether diffusion of NH3 down the bath-to-lumen NH3 concentration gradient could account for the measured ammonia secretion, we measured the permeabilities to NH3 (1.6 X 10(-2) cm/s) and NH4+ (4.5 X 10(-5) cm/s). From these values, we calculated predicted rates of passive NH3 secretion (-3.6 pmol X mm-1 X min-1) and passive NH4+ absorption (0.9 pmol X mm-1 X min-1). The predicted rate of net ammonia secretion exceeded the measured rate, indicating that passive NH3 secretion can fully account for the measured flux. In additional experiments, 10(-4) M acetazolamide in bath and perfusate inhibited net bicarbonate absorption by approximately 80%, but the rate of ammonia secretion was unaffected. CONCLUSIONS: S2 proximal straight tubules spontaneously secrete ammonia as required for generation of a corticomedullary ammonia concentration gradient by counter-current multiplication. Diffusion of NH3 down a concentration gradient created by luminal acidification can account for the ammonia secretion. A substantial passive lumen-to-bath backflux of NH4+ occurs. Acetazolamide does not inhibit ammonia secretion.

Acetazolamide↗
PubMed1987

Bicarbonate and ammonia transport in isolated perfused rat proximal straight tubules.

Bicarbonate, ammonia, and fluid transport were studied in isolated perfused proximal straight tubules from rats. The mean rate of fluid absorption (0.77 nl X min-1 X mm-1) and the mean rate of total CO2 absorption (42 pmol X min-1 X mm-1) exceeded corresponding rates measured previously in rabbit proximal straight tubules. The limiting total CO2 concentration when the tubules were perfused at slow flow rates was 5 mM, a value similar to those reported previously for rat proximal convoluted tubules and thick ascending limbs. When rat proximal straight tubules were perfused and bathed with solutions containing 1 mM total ammonia at slow perfusion rates, the measured total ammonia concentration in collected fluid rose to a level predicted by the diffusion trapping model of ammonia secretion in the absence of a luminal disequilibrium pH. We conclude the proximal straight tubule of the rat can absorb bicarbonate at a rate that can account for a large portion of the bicarbonate absorption measured in vivo between the late proximal convoluted tubule and the early distal tubule, the rat proximal straight tubule is capable of transepithelial ammonia secretion, most likely by NH3 diffusion down a concentration gradient generated by luminal acidification, and the rat proximal straight tubule apparently does not generate a luminal disequilibrium pH despite the occurrence of proton secretion, implying the presence of endogenous luminal carbonic anhydrase.

Ammonia↗
PubMed1986

Binding of 3H-phenamil, an irreversible amiloride analog, to toad urinary bladder: effects of aldosterone and vasopressin.

Phenamil, an analog of amiloride, has previously been shown to bind specifically to sodium channels in toad bladder (J.L. Garvin et al., J. Membrane Biol. 87:45-54, 1985). In this paper, 3H-phenamil was used to measure sodium channel density in both isolated epithelial cells and intact bladders. From the specific binding to intact bladders, a channel density of 455 +/- 102 channels/micron2 was calculated. No correlation between specific binding and the magnitude of irreversible inhibition of short-circuit current was found. Pretreatment of intact bladders with 1 mg/ml trypsin reduced specific binding to isolated cells by 82 +/- 5%. In isolated cells, neither aldosterone nor vasopressin had any significant effect on specific phenamil binding. It is inferred that phenamil binds to both open and closed channels which may be either in the mucosal membrane or in the submembrane space. Finally, and rather surprisingly, we found that 3H-phenamil binds irreversibly to the basolateral membrane at concentrations as low as 4 X 10(-7) M. Therefore, care must be used in interpreting binding studies with amiloride or its analog at such concentrations.

Aldosterone↗
PubMed1986

Epithelial responses of rabbit tongues and their involvement in taste transduction.

The response of rabbit tongue, placed in a modified Ussing chamber, to salts [KCl, NaCl, NH4Cl, tetraethylammonium chloride (TEACl)], sweeteners (D-glucose, sucrose), acid (HCl), and a bitter tastant, quinine, was investigated for the first time. These experiments were conducted to further explore the new paradigm of taste transduction that correlates changes in the electrical behavior of lingual epithelia with neural responses. In addition, the responses of rabbit tongue data were compared with previously published data on dog and frog tongues. When increases in short-circuit current (Isc) for rabbit tongue induced by hyperosmotic concentrations of several salts were compared, the following sequence was found: KCl greater than NH4Cl greater than NaCl greater than TEACl. These results are consistent with previously published integrated chorda tympani responses (ICTR) as well as behavioral studies that showed that rabbits prefer KCl to NaCl with their natural diet. Pharmacological studies using ouabain and amiloride suggest that K and Na are traversing rabbit tongue through different pathways. The maximal stimulation of Isc by D-glucose and sucrose (in 50 mM NaCl) was significantly smaller than those measured for KCl and NaCl. The rabbit tongue was sensitive to HCl at concentrations less than 1 mM in agreement with ICTR measurements.

Animals↗
PubMed1986

Spontaneous luminal disequilibrium pH in S3 proximal tubules. Role in ammonia and bicarbonate transport.

We determined whether a spontaneous luminal disequilibrium pH, pHdq (pH measured - pH equilibrium), was present in isolated perfused rabbit S2 and S3 proximal tubules. Luminal pH was measured by perfusing with the fluorescent pH probe 1,4-DHPN, and the equilibrium pH was calculated from the measured collected total CO2 and dissolved CO2 concentrations. S2 tubules failed to generate a spontaneous pHdq. S3 tubules generated a spontaneous acidic pHdq of -0.46 +/- 0.15 (P less than 0.05), which was obliterated following the addition of carbonic anhydrase (0.1 mg/ml) to the perfusate. In S3 tubules perfused and bathed in 4 mM total ammonia, luminal total ammonia rose from 4.08 +/- 0.05 mM (perfusate) to 4.95 +/- 0.20 mM (collected fluid) (P less than 0.02). Carbonic anhydrase added to the perfusate prevented the rise in the collected total ammonia concentration. We conclude that the rabbit S3 proximal tubule lacks functional luminal carbonic anhydrase. The acidic pHdq in the S3 segment enhances the diffusion of NH3 into the lumen. In contrast, the S2 segment has functional luminal carbonic anhydrase.

Ammonia↗
PubMed1985

Phenamil: an irreversible inhibitor of sodium channels in the toad urinary bladder.

Several new amiloride analogues and two reported photoaffinity analogues were tested for irreversible inhibition of short-circuit current, Isc, in toad bladder. Bromoamiloride, a photoaffinity analogue, induced 40% irreversible inhibition at 500 microM after irradiation with ultraviolet light greater than or equal to 320 nm. Iodoamiloride caused no irreversible inhibition. Of the new analogues tested, only 3,5-diamino-6-chloro-N-[(phenylamino) amino-methylene] pyrazinecarboxamide, phenamil, irreversibly inhibited Isc at concentrations of 0.05 to 5 microM when added to the mucosal solution. Irreversible inhibition of Isc by phenamil may be attributed to specific blockage of the mucosal sodium channels, which depended on: 1) time of exposure; 2) mucosal pH; 3) mucosal sodium concentration. For example, 5 microM phenamil irreversibly inhibited Isc by 38% in 103 mM Na at pH 8.6 and nearly 75% in 30 mM Na at pH 6.4 after a 40-min exposure. Irreversible inhibition occurred in two phases with time constants of less than or equal to 10 min and approximately 140 min. Due to its irreversible nature, phenamil may be used to measure channel density.

Amiloride↗
PubMed1985

Salt and acid studies on canine lingual epithelium.

A section of dog tongue just anterior to the circumvallate papillae was placed in an Ussing chamber and characterized with respect to monovalent cation transport pathways, response to HCl, and the interaction between salt and acid for the purposes of determining whether ion transport through the tongue is related to the transduction process. The primary findings of this work follow. The stimulation of the short-circuit current (Isc) with decreasing pH on the dorsal surface results from a net Cl- efflux through an amiloride- and ouabain-insensitive pathway that changed its selectivity from cationic at neutral pH to anionic at lower pH values. The stimulation is abolished at isotonic NaCl concentrations. This behavior would not be predicted from the proton-binding model to a "sour receptor" as suggested by Beidler (Handbook of Sensory Physiology; Berlin: Springer-Verlag, 1971). In symmetrical solutions of Krebs-Henseleit buffer, Isc is inhibited 30% by 10(-4) M amiloride on the mucosal side and 100% by 10(-3) M ouabain on the serosal side. These results coupled with net Cl- and Na+ flux measurements show that Isc is due to a net Cl- efflux. With 1.0 M NaCl on the dorsal side, 10(-4) M amiloride inhibits Isc 84%. In contrast, when the mucosal surface is bathed in KCl (1.0 M), CsCl (1.0 M), or HCl (10(-3) M), Isc is insensitive to both 10(-4) M amiloride and/or 10(-3) M ouabain. These data suggest that K+, Cs+, and H+ traverse the tongue through different pathways from Na+ and are not energized by a Na+ gradient.

Amiloride↗