Cobern E. Ott

Effect of Chloride on Renin and Blood Pressure Responses to Sodium Chloride

Both the inhibition of renin release by sodium chloride and salt-sensitive hypertension have been attributed to sodium. We evaluated the contribution of chloride to these responses to sodium chloride. In the Sprague-Dawley rat, acute and chronic administration of sodium salts other than sodium chloride failed to suppress plasma renin activity, whereas renin was inhibited by both sodium chloride and by selective chloride (without sodium) loading. Plasma renin activity was stimulated by selective chloride depletion. Similarly, in humans, plasma renin activity was suppressed by sodium chloride but not by sodium bicarbonate infusion. In a preliminary study in the Dahl salt-sensitive rat, in contrast to sodium chloride loading, sodium bicarbonate loading failed to produce hypertension. Thus, both the renin and possibly the blood pressure responses to sodium chloride are dependent on chloride.

Guanylyl cyclase-C receptor mRNA distribution along the rat nephron.

Guanylin (GN) and uroguanylin (UGN) are two recently identified peptides that have been shown to affect water and electrolyte transport in both the intestine and the kidney. Mechanistically, the effects of both peptides are thought to be mediated by intracellular cGMP which results from ligand binding to a plasma membrane guanylyl cyclase-C (GC-C) receptor. To date, the specific intrarenal site(s) of GN and UGN action have not been established. To begin to address this issue, the present studies utilized semi-quantitative RT-PCR to assess the distribution of GC-C mRNA in specific microdissected segments of the rat nephron. GC-C mRNA expression was highest in the cortical collecting tubule, followed by the proximal convoluted tubule, medullary thick ascending limb and collecting tubule, and thin limbs of Henles loop. Expression levels were significantly lower in all other segments tested, including the glomerulus. The renal tubular expression pattern for cGMP-dependent protein kinase II (cGK-II) mRNA, which is activated in response to GN/UGN-dependent cGMP accumulation, was similar to that for GC-C. Notably, both GN and UGN mRNAs were also expressed along the nephron. The highest levels of expression for both peptides were detected in the medullary collecting tubule. Lower, but comparable levels of GN and UGN expression also occurred in the cortical collecting tubule, cortical and medullary thick ascending limb, and thin limbs of Henles loop. In the proximal convoluted tubule, GN mRNA expression was also quite high, while UGN mRNA was almost undetectable. The presence of renal GC-C and cGK-II in the kidney are consistent with a proposed endocrine function for GN and UGN. In addition however, the present data suggest that intrarenally synthesized GN and UGN may also contribute to the regulation of renal tubular transport.

Mechanism of atrial natriuretic peptide release with increased inspiratory resistance

Elevated plasma atrial natriuretic peptide (ANP) levels and concomitant increases in renal sodium and water excretion are often encountered in respiratory diseases associated with increased airway resistance such as obstructive sleep apnea. The present study utilized an anesthetized rat model to determine the principal mechanism(s) responsible for stimulation of ANP release in this clinical syndrome. A 10-minute increase in external resistive loading, which reduced peak tracheal pressure to -15 to -17 mm Hg produced a significant increase in both central venous pressure and right atrial transmural pressure. This maneuver subsequently resulted in significant transient increases in glomerular filtration rate; urine flow; urinary Na+, K+, and Cl- excretion; and urinary cyclic guanosine monophosphate (cGMP) excretion, which was taken as an index of increased circulating levels of ANP. Similar changes in renal function and cGMP excretion occurred when arterial PO2 was lowered to a degree equivalent to that seen with increased resistive loading. Lowering arterial PO2 also significantly increased mean central venous pressure and right atrial transmural pressure. Conversely, the resistive loading-induced changes in renal function and cGMP excretion did not occur when the reduction in arterial PO2 was prevented by breathing a high O2 gas mixture during the resistive loading. Additionally, O2 supplementation prevented the increases in both mean central venous pressure and right atrial transmural pressure caused by increased resistive loading. These data indicate that the elevated ANP release that results from an acute increase in external resistive loading is not caused by a decrease in intrathoracic pressure but rather suggest that the elevated ANP release is primarily caused by an increased right atrial transmural pressure resulting from hypoxia-induced pulmonary vasoconstriction.

P450 arachidonate metabolites mediate bradykinin-dependent inhibition of NaCl transport in the rat thick ascending limb.

Recent studies from this laboratory demonstrated that bradykinin transiently elevates intracellular Ca2+ and inhibits Cl-reabsorption in the in vitro microperfused medullary thick ascending limb (mTAL) of the rat. The present study was designed to identify the intracellular signaling mechanism(s) that mediate this response. Preincubation with the intracellular calcium chelator BAPTA (10(-5) M) completely eliminated the bradykinin-dependent increase in intracellular Ca2+ and the suppression of Cl- transport. Preincubation with the cGMP-dependent protein kinase inhibitor H-89 (10(-5) M) had no effect on the transport response to bradykinin. In contrast, 17-octadecynoic acid (17-ODYA; 10(-5) M), a suicide-substrate inhibitor of renal cytochrome P450 omega-hydroxylase, completely blocked the transport response to bradykinin, while the cyclooxygenase inhibitor sodium meclofenamate (10(-5) M) had no effect. Finally, addition of the cytochrome P450 omega-hydroxylase metabolite 20-hydroxyeicosatetraenoic acid (20-HETE; 10(-8) M) to the bathing medium significantly inhibited Cl- transport in the mTAL (delta -39 +/- 6.0%; p < 0.05), while the epoxygenase metabolite 5,6-epoxyeicosatrienoic acid (5,6-EET; 10(-8) M) had no effect. These data suggest that the bradykinin-dependent inhibition of Cl- transport in the mTAL of the rat is mediated by cytochrome P450 dependent metabolite(s) of arachidonic acid.

Site-specific effects of dietary salt intake on guanylin and uroguanylin mRNA expression in rat intestine

Guanylin and uroguanylin are newly discovered intestinal peptides that have been shown to affect NaCl transport in both the intestine and kidney. The present study tests the hypothesis that guanylin and uroguanylin mRNA expression in each major region of the intestine is regulated by NaCl intake. Semiquantitative multiplex RT-PCR analysis was used to determine the molecular expression of guanylin and uroguanylin in the duodenum, jejunum, ileum, and colon in rats maintained on low (LS), normal (NS), or high (HS) NaCl intake for 4 days. LS intake reduced the expression of uroguanylin, and to a lesser degree, guanylin mRNA in all intestinal segments compared to NS intake. The duodenum was the site of the greatest decrease for both. In contrast, HS intake significantly increased the expression of guanylin mRNA only in the duodenum and jejunum and had minimal effect on uroguanylin mRNA. The minimum time required for altered gene expression was determined by delivering an oral NaCl challenge directly to the gastrointestinal tract by oro-gastric administration to LS or NS animals. In LS rats, NaCl oro-gastric administration significantly increased mRNA expression of both peptides in all intestinal segments. Furthermore, the increases in guanylin and uroguanylin mRNA were detected within 4 h and plateaued by 8 h. Conversely, acute oro-gastric administration of the same NaCl solution to NS rats caused elevations of guanylin mRNA only in the duodenum and jejunum, and of uroguanylin mRNA only in the ileum and colon. In conclusion, the data demonstrate that variations in NaCl intake lead to intestinal segment-specific changes in guanylin and uroguanylin mRNA expression.

Failure of salt loading to inhibit tissue norepinephrine turnover in prehypertensive Dahl salt-sensitive rats.

To determine if alterations of electrolyte balance or sympathetic nervous system activity are present in Dahl salt-sensitive rats (DS) before the onset of hypertension, we compared electrolyte balances, extracellular fluid volume (inulin space), plasma volume (radiolabeled albumin), and norepinephrine turnover in peripheral tissues (heart and interscapular brown fat) in prehypertensive DS and Dahl salt-resistant rats (DR). Animals were maintained for 5 to 7 days on either a “normal” or high NaCl diet. Tissue norepinephrine turnover was evaluated by measuring the rate at whkh norepinephrine content decreased following tyrosine hydroxylase inhibition with α-methyl-p-tvrosine. Blood pressure was higher (p < 0.05) in DS (135 ± 2 [SE] mm Hg) than in DR (129 ± 2 mm Hg) and was not affected by the diets. Extracellular fluid volume and net Naplus; and Clminus; balances did not differ between DS and DR. However, plasma volume was greater in DS than in DR (p < 0.05). In both fat and heart, norepinephrine turnover was decreased by dietary NaCl loading in DR (p < 0.01), but not in DS. Thus, the tendency of the DS to become hypertensive with high NaCl intake may be related to the combined effects of an increased plasma volume and the failure of high dietary NaCl to inhibit peripheral sympathetic nervous system activity.

Dopamine D1 receptor-dependent inhibition of NaCl transport in the rat thick ascending limb: mechanism of action.

Our previous in vitro microperfusion studies established that dopamine inhibits sodium chloride transport in the rat medullary thick ascending limb. The present study was designed to determine the intracellular signaling pathway mediating this response. The dopamine D1 receptor agonist fenoldopam (1 microM) inhibited sodium chloride transport in the thick ascending limb by 42+/-5%. The dopamine D1 receptor antagonist R-(+)-7-Chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine-HCl (SCH-23390) completely blocked this effect of fenoldopam. Suppression of protein kinase A activity using either myristoylated protein kinase inhibitor (PKI) or N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide.2HCl (H-89), as well as suppression of phospholipase C activity using 1-(6-((17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U-73122), had no effect on fenoldopam-dependent inhibition of transport. In contrast, inhibition of phospholipase A2 activity using E-6-(Bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (HELSS) significantly attenuated the effect of fenoldopam by 74%. The cytochrome P-450 monooxygenase inhibitor 17-octadecynoic acid (17-ODYA) and the protein kinase C inhibitor staurosporine both significantly attenuated the effects of fenoldopam by 67%. Exposure to 20-Hydroxy-(5Z, 8Z, 11Z, 14Z)-eicosatetraenoic acid (20-HETE) inhibited transport by 31+/-5%, and this effect was significantly attenuated by 66% in the presence of staurosporine. We propose a signaling pathway in which dopamine activates a calcium-independent phospholipase A2 in the medullary thick ascending limb. Released arachidonic acid is then metabolized to 20-HETE which subsequently increases protein kinase C activity that acts as a final transport effector.

Effect of luminal vasopressin on NaCl transport in the medullary thick ascending limb of the rat

The aim of the present study was to determine whether vasopressin affects NaCl reabsorption in the medullary thick ascending limb of the loop of Henle when administered selectively to the luminal membrane. At 5 x 10(-6) M and 10(-8) M, luminal [Arg8]vasopressin significantly inhibited Cl- transport in the in vitro microperfused rat medullary thick ascending limb by 46.4 +/- 5.9% (P < 0.01) and 32.4 +/- 2.0% (P < 0.05) respectively. The response to 10(-8) M luminal [Arg8]vasopressin was completely blocked by the vasopressin V1 receptor antagonist [beta-mercapto-beta,beta-cyclopenta-methylenepropionyl1, O-Me-Tyr2,Arg8]vasopressin (10(-6) M), and was mimicked by the vasopressin V1 receptor agonist [Phe1, Ile5, Orn8]vasopressin (10(-8) M; delta -35.0 +/- 4.5%; P < 0.05). Luminal administration of the vasopressin V2 receptor agonist [deamino-Cys1, D-Arg8]vasopressin (5 x 10(-6) M) had no effect on transport. These data suggest that luminal vasopressin can inhibit NaCl transport in the medullary thick ascending limb of the rat via vasopressin V1 receptors.

Modulation of hypoglycemia-induced increases in plasma epinephrine by estrogen in the female rat

Clinical studies have demonstrated that estrogen replacement therapy suppresses stress‐induced increases in plasma catecholamines. The present study determined whether normal circulating levels of estrogen can modulate hypoglycemia‐induced increases in plasma epinephrine (EPI). In anesthetized female rats, insulin‐induced hypoglycemia (0.25 U/kg) increased plasma EPI concentration to a significantly greater extent in 14‐day ovariectomized (OVEX) rats compared to that in sham‐operated controls. In 17β‐estradiol (E2)‐replaced OVEX rats, the hypoglycemia‐induced rise in plasma EPI was reduced significantly when compared to that in vehicle‐replaced OVEX rats. OVEX and E2 replacement had no effect on tyrosine hydroxylase or phenylethanolamine N‐methyltransferase mRNA levels in the adrenal medulla. In isolated adrenal medullary chromaffin cells, agonist‐induced increases in intracellular Ca2+ were unaffected by 48‐hr exposure to 10 nM E2. In contrast, acute (3‐min) exposure to micromolar concentrations of E2 dose‐dependently and reversibly inhibited agonist‐induced Ca2+ transients. In addition, in OVEX rats, a constant infusion of E2 significantly reduced the insulin‐induced increase in plasma EPI concentration compared to that in vehicle‐infused controls. These data demonstrate that physiologic levels of circulating E2 can modulate hypoglycemia‐induced increases in plasma EPI. This effect seems independent of steroid influence on adrenal medullary secretion or biosynthesis. In contrast, acute exposure to high levels of E2 can also suppress hypoglycemia‐induced increases in plasma epinephrine, due at least in part to inhibition of stimulus‐secretion coupling.

Acute effects of gentamicin on thick ascending limb function in the rat

It is well established that the aminoglycoside antibiotics can adversely affect proximal tubule function. Predominantly indirect evidence suggests that aminoglycosides may also affect function of more distal nephron segments. The present study utilized whole kidney clearance, in vivo micropuncture and in vitro microperfusion to directly determine whether acute gentamicin treatment affects sodium chloride transport in the thick ascending limb of the loop of Henle. Gentamicin (25 mg/kg) significantly increased urine flow, as well as sodium, potassium and chloride excretion within 15 min of intravenous injection. Glomerular filtration rate and proximal tubule fluid reabsorption were not altered by acute gentamicin treatment. In contrast, both fractional and absolute loop chloride transport was significantly decreased. In the in vitro microperfused medullary thick ascending limb, luminal but not basolateral administration of gentamicin (1 mM) significantly decreased chloride reabsorption when compared to time controls. These data suggest that the increased urine and electrolyte excretion associated with acute gentamicin treatment is, at least in part, a consequence of decreased transport in the thick ascending limb of Henles loop.