Deoxycorticosterone Pivalate Research Articles

Abstract P314: Sex-specific Role For The Circadian Clock Protein PER1 In Aortic Stiffness

Background: Increased aortic stiffness is independently associated with an increased risk of cardiovascular events and improvement in aortic stiffness positively correlates with better survival. Period 1 (PER1) is a core circadian clock protein and previous studies have shown that global or kidney-specific knockout (KO) of PER1 results in salt-sensitive hypertension, but the role of PER1 in the vasculature remains unknown. The goal of the present study was to test the hypothesis that loss of PER1 would lead to increased aortic stiffness. Methods: Male and female C57BL/6J (16-18 weeks old) mice with global or vascular smooth muscle cell (VSMC)-specific PER1 KO (n=7-10) and control mice (WT) were subjected to echocardiographic measurement of pulse wave velocity (PWV) to evaluate aortic stiffness. We further investigated if treatment with high salt diet plus an aldosterone analog (HS-DOCP, deoxycorticosterone pivalate) has any additional effect on aortic stiffness in male VSMCs-specific PER1 KO mice. Results: In mice on a normal diet, global KO of PER1 resulted in increased PWV in males (WT: 202±8, PER1 KO: 287±27 cm/s, p<0.01) but not in female mice (WT:219±14, PER1 KO: 225 ± 23 cm/s, p<0.80) compared to age-matched wild type. Further, morphometric analysis of the aorta showed increased adventitia thickness in male global PER1 KO mice (WT: 41.7±2.3, PER1 KO: 54.6±2.4 um, (p<0.01). These data led us to generate VSMC PER1 KO mice by crossing Tagln Cre mice with floxed PER1 mice. VSMC KO was confirmed by recombination PCR. PWV was not different between male VSMC PER1 KO and Cre negative littermate control (CNTL) mice on normal diet, however, it was increased in VSMC PER1 KO mice after treatment with HS-DOCP (CNTL: HS-DOCP: 241 ±12, PER1 KO: HS-DOCP: 307±18 cm/s, p<0.01). PWV in female VSMCs PER1 KO mice was not statistically significant compared to littermate CNTL mice (CNTL: 247±12, VSMC PER1 KO: 233 ± 25 cm/s, p<0.50). Conclusion: Our findings indicate that loss of the clock protein PER1 increases aortic stiffness in male but not in female mice suggesting a sex specific role for the circadian clock in the pathology of aortic stiffness. Further studies are warranted to identify the potential mechanisms involved in the pathogenesis of PER1-dependent aortic stiffness.

Pivalato de desoxicorticosterona no controle do hipoadrenocorticismo canino – relato de casos

Primary hypoadrenocorticism or Addison’s disease is an endocrinopathy involving glucocorticoid deficiency, commonly associated with mineralocorticoid deficiency. The most commonly used treatment for hormone replacement involves the combination of fludrocortisone and prednisone. Deoxycorticosterone pivalate (DOCP) has been shown in the literature to be superior in electrolyte control in patients with hypoadrenocorticism, and should always be associated with prednisone/prednisolona. In this study, we report the benefit of DOCP and the two-stage dose adjustment protocol for this drug in 2 different cases. There was superior efficacy in electrolyte control and reduction of side effects with the DOCP and prednisolone protocol compared to the use of fludrocortisone in both patients.

Use of Deoxycorticosterone Pivalate by Veterinarians: A Western European Survey.

This study aims to gather knowledge about the use of deoxycorticosterone pivalate (DOCP) by Western European Veterinarians (WEV) in dogs with typical hypoadrenocorticism. An observational cross-sectional study was conducted using an online survey, translated into four languages and disseminated to veterinary affiliates and mailing lists in six countries of Western Continental Europe. Respondents were tasked to share their therapeutic approach to hypoadrenocorticism, whether they preferred DOCP or fludrocortisone and the specific practical use of DOCP. One-hundred and eighty-four responses were included. Of these, 79.9% indicated that they preferred prescribing DOCP over fludrocortisone as a first-line treatment for mineralocorticoid supplementation. A total of 154 respondents had used DOCP at least once. Eighty percent of those who reported their initial dosage prescribed 2.2 mg/kg. After starting DOCP, 68.2% of the respondents assess electrolytes 10 and 25 days after administration following manufacturer instructions. In stable dogs, electrolytes are monitored quarterly, monthly, semi-annually, and annually by 44.2%, 34.4%, 16.9%, and 4.6% of respondents respectively. When treatment adjustment is required, 53% prefer to reduce dosage while 47% increase the interval between doses. Overall, DOCP is the preferred mineralocorticoid supplementation among WEV. Reported variability underlies the need to investigate the best strategies for DOCP use and therapeutic adjustments.

Knockout of Per1 Exacerbates the Hypertensive Phenotype of the Dahl Salt Sensitive Rat

The dynamic between blood pressure, its circadian rhythm, and the circadian clock genes has been considered as a factor in the subsequent renal injury that accompanies hypertension. Various circadian clock proteins such as BMAL1 and PERIOD (PER) function in a marriage of positive and negative feedback loops that control transcriptional regulation. Of particular interest is PER1 which naturally acts in the negative loop of the cycle as a repressor, but recent findings show that it may have additional roles as a transcription activator. PER1 has a role in renal sodium handling as a target of aldosterone, and it has been linked to the expression of the α‐subunit of the epithelial sodium channel (α‐ENaC). Investigators have found that knockout of PER1 in 129/sv mice causes a decrease in mean arterial pressure (MAP); however, when the gene is knocked out in the C57BL/6J mouse on a 4% NaCl diet (high salt, HS) with deoxycorticosterone pivalate (DOCP) treatment, there is a significant increase in MAP with animals displaying a “non‐dipping” pattern of MAP. The wildtype mice that were on HS with DOCP treatment had a 11.4 ± 1.9% decrease in MAP from night to day whereas the knockout mice with the same conditions had only a 5.7 ± 1.4% decrease. To test the contribution of PER1 in the development of salt‐induced hypertension, we created the Per1 knockout rat on the Dahl Salt‐Sensitive rat background (SS; knock out SSPer1−/−). Eight to 9‐week old SS and SSPer1−/− male rats were implanted with DSI telemetry units to measure blood pressure on a 4% NaCl diet (high salt, HS) for 3 weeks. MAP in SSPer1−/− rats was significantly higher compared to wild type SS rats (191 ± 7 vs. 168 ± 4 mmHg, P = 0.031; respectively) after 3 weeks of HS. We also revealed a significant increase in albuminuria in SSPer1−/− rats on HS compared to SS rats on HS (0.10 ± 0.01 vs. 0.05 ± 0.01 Alb/Cre Clearance ratio, P = 0.027; respectively). Knockout of Per1 in SS rats on HS also resulted in a significant increase in cholesterol (200.0 ± 11.8 vs. 135.0 ± 9.7 mg/Dl, P = 0.003; respectively) and alkaline phosphatase (37.5 ± 3.6 vs. 77.6 ± 3.3 U/L, P = <0.001; respectively). Furthermore, SSPer1−/− rats after 3 weeks of HS diet had significant decreases in creatinine clearance compared to SS rats (65 ± 6 vs. 127 ± 24 mL/hour, P = 0.036; respectively). Here, we show that lack of PER1 causes a dramatization of the salt‐sensitive phenotype of the SS rat with a decline in renal function. These data propose that the role PER1 plays in sodium homeostasis in the kidney is pivotal for proper blood pressure regulation.Support or Funding InformationNational Institutes of Health Grants R35 HL135749 (AS) and R01 DK109570 (MLG) and by the American Heart Association 18PRE34030127 (DS)

Female C57BL/6J mice lacking the circadian clock protein PER1 are protected from nondipping hypertension.

The circadian clock is integral to the maintenance of daily rhythms of many physiological outputs, including blood pressure. Our laboratory has previously demonstrated the importance of the clock protein period 1 (PER1) in blood pressure regulation in male mice. Briefly, a high-salt diet (HS; 4% NaCl) plus injection with the long-acting mineralocorticoid deoxycorticosterone pivalate (DOCP) resulted in nondipping hypertension [<10% difference between night and day blood pressure (BP) in Per1-knockout (KO) mice but not in wild-type (WT) mice]. To date, there have been no studies that have examined the effect of a core circadian gene KO on BP rhythms in female mice. The goal of the present study was to determine whether female Per1-KO mice develop nondipping hypertension in response to HS/DOCP treatment. For the first time, we demonstrate that loss of the circadian clock protein PER1 in female mice does not significantly change mean arterial pressure (MAP) or the BP rhythm relative to female C57BL/6 WT control mice. Both WT and Per1-KO female mice experienced a significant increase in MAP in response to HS/DOCP. Importantly, however, both genotypes maintained a >10% dip in BP on HS/DOCP. This effect is distinct from the nondipping hypertension seen in male Per1-KO mice, demonstrating that the female sex appears to be protective against PER1-mediated nondipping hypertension in response to HS/DOCP. Together, these data suggest that PER1 acts in a sex-dependent manner in the regulation of cardiovascular rhythms.

Desoxycorticosterone pivalate-salt treatment leads to non-dipping hypertension in Per1 knockout mice.

Increasing evidence demonstrates that circadian clock proteins are important regulators of physiological functions including blood pressure. An established risk factor for developing cardiovascular disease is the absence of a blood pressure dip during the inactive period. The goal of the present study was to determine the effects of a high salt diet plus mineralocorticoid on PER1-mediated blood pressure regulation in a salt-resistant, normotensive mouse model, C57BL/6J. Blood pressure was measured using radiotelemetry. After control diet, wild-type (WT) and Per1 (KO) knockout mice were given a high salt diet (4% NaCl) and the long-acting mineralocorticoid deoxycorticosterone pivalate. Blood pressure and activity rhythms were analysed to evaluate changes over time. Blood pressure in WT mice was not affected by a high salt diet plus mineralocorticoid. In contrast, Per1 KO mice exhibited significantly increased mean arterial pressure (MAP) in response to a high salt diet plus mineralocorticoid. The inactive/active phase ratio of MAP in WT mice was unchanged by high salt plus mineralocorticoid treatment. Importantly, this treatment caused Per1 KO mice to lose the expected decrease or 'dip' in blood pressure during the inactive compared to the active phase. Loss of PER1 increased sensitivity to the high salt plus mineralocorticoid treatment. It also resulted in a non-dipper phenotype in this model of salt-sensitive hypertension and provides a unique model of non-dipping. Together, these data support an important role for the circadian clock protein PER1 in the modulation of blood pressure in a high salt/mineralocorticoid model of hypertension.

Abstract 130: Modulation of Salt and Mineralocorticoid Sensitivity of Blood Pressure by the Circadian Clock Protein Per1

The circadian clock is important for maintaining rhythms in physiological functions including blood pressure (BP). Circadian disruption leads to increased disease risk. The clock has also been implicated in the maintenance of a normal dip in BP at night. In humans, non-dipping (night/day difference in BP&lt;10%) is associated with an increased risk of cardiovascular and kidney disease. Dipping status can also be affected by salt intake and by hormones such as the mineralocorticoid aldosterone. The goal of this study was to determine the effects of a high salt (HS, 4% NaCl) diet plus mineralocorticoid (deoxycorticosterone pivalate (DOCP)) on BP regulation by the circadian clock protein Per1 in C57BL/6J mice. BP was monitored in conscious, unrestrained male mice by radiotelemetry and values are reported as mean arterial pressure (MAP) ± SEM. Under control conditions, MAP in male WT mice was 112.5 ± 1.08 mmHg during the night when mice are active and decreased to 102.1 ± 1.7 mmHg during the day, a “dip” in MAP of 9.2 ± 1.3%. Similarly, Per1 KO mice dip 14 ± 1.4%, with night time MAP of 119.8 ± .9 mmHg which decreased to 103 ± 1.4 mmHg during the day. On HS/DOCP, WT mice MAP decreased from 114.5 ± 1.1 mmHg to 101.5 ± 1.92 mmHg (night indicated by shaded bars in figure). This 11.4 ± 1.9% dip in WT mice was not significantly different from what was observed under control conditions. In contrast, Per1 KO mice display a significantly attenuated dip of 5.7 ± 1.4% with night time MAP of 125.3 ± 1.5 mmHg dropping to 118.1 ± 1 mmHg during the inactive day period (p&lt;0.05). Thus, HS/DOCP treatment in Per1 KO mice leads to non-dipping hypertension. This is the first report of this phenotype in a single clock gene KO.

Mineralocorticoids stimulate the activity and expression of renal H,K‐ATPases

Mineralocorticoids such as deoxycorticosterone activate Na+ reabsorption and H+ secretion in the renal collecting duct. In this study, the effect of deoxycorticosterone pivalate (DOCP) on renal H+,K+‐ATPase activity and expression was investigated. Eight days after female wild type (WT) mice received DOCP, collecting ducts were perfused in vitro to measure H+,K+‐ATPase‐dependent H+ secretion after an acute intracellular acid load. DOCP stimulated H+,K+‐ATPase mediated H+ secretion in A type intercalated cells of inner cortical collecting ducts by 70% whereas no effect of DOCP was observed in B type intercalated cells compared to control. DOCP dramatically increased HKα2 mRNA expression in outer and inner medulla (246% and 1040%, respectively) and did not affect HKα1 mRNA expression. DOCP treatment caused significant body weight gain in WT mice and had little effect on body weight in mice lacking both α subunits (HKα1,2−/−). DOCP caused a similar degree of hypokalemia in WT and HKα1,2−/− mice. Importantly, WT developed metabolic alkalosis whereas HKα1,2−/− mice did not. In summary, these data clearly demonstrate a physiological role for renal H+,K+‐ATPases in mineralocorticoid stimulated H+ secretion and suggest a role for these enzymes in Na+ reabsorption.Studies were funded by NIH RO1‐DK‐049750 to C.S.W and 5T32DK007518‐22 to M.M.G.

Deoxycorticosterone upregulates PDS (Slc26a4) in mouse kidney: role of pendrin in mineralocorticoid-induced hypertension.

Pendrin is an anion exchanger expressed along the apical plasma membrane and apical cytoplasmic vesicles of type B and of non-A, non-B intercalated cells of the distal convoluted tubule, connecting tubule, and cortical collecting duct. Thus, Pds (Slc26a4) is a candidate gene for the putative apical anion-exchange process of the type B intercalated cell. Because apical anion exchange-mediated transport is upregulated with deoxycorticosterone pivalate (DOCP), we tested whether Pds mRNA and protein expression in mouse kidney were upregulated after administration of this aldosterone analogue by using quantitative real-time polymerase chain reaction as well as light and electron microscopic immunolocalization. In kidneys from DOCP-treated mice, Pds mRNA increased 60%, whereas pendrin protein expression in the apical plasma membrane increased 2-fold in non-A, non-B intercalated cells and increased 6-fold in type B cells. Because pendrin transports HCO3- and Cl-, we tested whether DOCP treatment unmasks abnormalities in acid-base or NaCl balance in Pds (-/-) mice. In the absence of DOCP, arterial pH, systolic blood pressure, and body weight were similar in Pds (+/+) and Pds (-/-) mice. After DOCP treatment, weight gain and hypertension were observed in Pds (+/+) but not in Pds (-/-) mice. Moreover, after DOCP administration, metabolic alkalosis was more severe in Pds (-/-) than Pds (+/+) mice. We conclude that pendrin is upregulated with aldosterone analogues and is critical in the pathogenesis of mineralocorticoid-induced hypertension and metabolic alkalosis.

ANG II reduces net acid secretion in rat outer medullary collecting duct.

In rat outer medullary collecting duct (OMCD), the mechanism(s) and regulation of H+ secretion are not understood fully. The effect of changes in acid-base balance and the renin-angiotensin system on net H+ secretion was explored. Rats received NaCl, NaHCO3, NH4Cl, or nothing in their drinking water for 7 days. Total ammonia and total CO2 (JtCO2) fluxes were measured in OMCD tubules perfused in vitro from rats in each treatment group. JtCO2 was reduced in tubules from rats drinking NH4Cl relative to those drinking NaHCO3. Because NH4Cl intake increases plasma renin and aldosterone, we asked if upregulation of the renin-angiotensin system reduces net H+ secretion. Deoxycorticosterone pivalate administered in vivo did not affect JtCO2. However, ANG II given in vivo at 0.1 ng/min reduced JtCO2 by 35%. To determine if ANG II has a direct effect on acid secretion, JtCO2 was measured with ANG II applied in vitro. ANG II (10-8 M) present in the bath solution reduced JtCO2 by 35%. This ANG II effect was not observed in the presence of the AT1 receptor blocker candesartan. In conclusion, in rat OMCD, JtCO2 is paradoxically reduced with NH4Cl ingestion. Increased circulating ANG II, as occurs during metabolic acidosis, reduces JtCO2.

Contribution of the Na+-K+-2Cl− Cotransporter (NKCC1) to Transepithelial Transport of H+, NH4 +, K+, and Na+ in Rat Outer Medullary Collecting Duct

ABSTRACT. In rat kidney, the “secretory” isoform of the Na-K-Cl cotransporter, NKCC1 (BSC-2), localizes to the basolateral membrane of the α intercalated cell, the acid secreting cell of the outer medullary collecting duct (OMCD). This laboratory has reported that NKCC1 mediates Cl− uptake across the basolateral membrane in series with Cl− secretion across the apical membrane in rat OMCD. NKCC1 transports NH4+, K+, and Na+ as well as Cl−; therefore, a role for the cotransporter in the process of HCl, NH4Cl, KCl, and NaCl secretion has been suggested. Thus, it was determined if bumetanide, an inhibitor of NKCC1, alters transepithelial cation transport in rat OMCD. OMCD tubules from deoxycorticosterone pivalate (DOCP)–treated rats were perfused in vitro. Hydration of CO2, rather than NH4+, provides the principle source of H+ for net acid secretion. In HCO3−/CO2-buffered solutions, no effect of bumetanide on net K+ flux was detected. Under some conditions, bumetanide addition resulted in a small reduction in secretion of net H+ equivalents. Transepithelial Na+ flux, JNa, was −1.5 ± 1.7 pmol/mm per min, values not different from zero. However, with the application of bumetanide to the bath, JNa was +5.2 ± 1.3 pmol/mm per min (P &lt; 0.05), which indicates net Na+ absorption. In conclusion, inhibition of NKCC1 in rat OMCD changes transepithelial movement of Na+ and Cl−. The role of NKCC1 in the secretion of net H+ equivalents is small.

Contribution of the Na+-K+-2Cl- cotransporter NKCC1 to Cl- secretion in rat OMCD.

In rat kidney the "secretory" isoform of the Na+-K+-2Cl- cotransporter (NKCC1) localizes to the basolateral membrane of the alpha-intercalated cell. The purpose of this study was to determine whether rat outer medullary collecting duct (OMCD) secretes Cl- and whether transepithelial Cl- transport occurs, in part, through Cl- uptake across the basolateral membrane mediated by NKCC1 in series with Cl- efflux across the apical membrane. OMCD tubules from rats treated with deoxycorticosterone pivalate were perfused in vitro in symmetrical HCO/CO2-buffered solutions. Cl- secretion was observed in this segment, accompanied by a lumen positive transepithelial potential. Bumetanide (100 microM), when added to the bath, reduced Cl- secretion by 78%, although the lumen positive transepithelial potential and fluid flux were unchanged. Bumetanide-sensitive Cl- secretion was dependent on extracellular Na+ and either K+ or NH, consistent with the ion dependency of NKCC1-mediated Cl- transport. In conclusion, OMCD tubules from deoxycorticosterone pivalate-treated rats secrete Cl- into the luminal fluid through NKCC1-mediated Cl- uptake across the basolateral membrane in series with Cl- efflux across the apical membrane. The physiological role of NKCC1-mediated Cl- uptake remains to be determined. However, the role of NKCC1 in the process of fluid secretion could not be demonstrated.

Inhibition by epinephrine of AVP- and cAMP-stimulated Na+ and water transport in Dahl rat CCD.

We examined the effects of epinephrine in perfused cortical collecting ducts (CCD) isolated from inbred Dahl-Rapp salt-sensitive (SS) and salt-resistant (SR) rats and from Sprague-Dawley (SD) rats. Rats were treated with 2.5 mg deoxycorticosterone pivalate (DOC; depot injection 4-9 days before study), and the CCD were treated with 220 pM vasopressin (AVP) to maximize Na+ transport. In CCD from all three strains 10 microM epinephrine in the bathing solution completely inhibited net Na+ transport, osmotic water permeability (Pf), and transepithelial voltage. In the SS CCD, epinephrine increased the fractional resistance of the luminal membrane to the same extent as 10 microM amiloride, indicating that it blocked the amiloride-sensitive conductance of the luminal membrane. Even at 100 nM epinephrine inhibited 80-100% of Na+ and water transport, and 1 microM yohimbine reversed or prevented these effects. In SS CCD, 0.1 mM 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) plus 0.1 mM 3-isobutyl-1-methylxanthine in place of AVP increased lumen-to-bath Na+ flux (J1-->b) from 56 +/- 5 to 143 +/- 3 pmol.min-1 x mm-1 and Pf from 6 +/- 12 to 1067 +/- 152 microns/s, but 100 mM epinephrine still significantly inhibited cAMP-stimulated J1-->b and Pf by 40 +/- 5% and 31 +/- 9%, respectively. Similar results were observed in the SR and SD rat CCD; however, the ability of yohimbine to reverse the epinephrine effect on cAMP-dependent transport was variable among the rat strains. We conclude that epinephrine acts via an alpha 2-receptor to inhibit adenylate cyclase but that at least one additional intracellular second messenger system may be involved.

Effects of ET-1 on water and chloride transport in cortical collecting ducts of the rat.

Endothelin-1 (ET-1) is known as a vasoconstrictor peptide. However, recent reports suggested the effects on the transport of renal tubule. We previously reported that ET-1 inhibited arginine vasopressin (AVP)-dependent adenosine 3',5'-cyclic monophosphate in rat collecting ducts. Physiologically, ET-1 reversibly and significantly inhibited AVP-stimulated water permeability in inner medullary collecting duct (IMCD). We therefore investigated the effects on water and electrolyte transport in rat cortical collecting ducts (CCD), where Na and Cl are actively reabsorbed more than in IMCD. Pathogen-free male Sprague-Dawley rats weighing 80-120 g were used after treatment with deoxycorticosterone pivalate for 1-2 wk. Isolated CCD were microperfused in vitro. The Cl concentration was measured by a continuous-flow ultra-microcolorimeter, and the raffinose concentration was measured as a volume marker by a continuous-flow ultra-microfluorometer. In the presence of 10(-9) M AVP, 10(-8) M ET-1 significantly inhibited fluid absorption (nl.mm-1 x min-1) from 0.25 +/- 0.02 to 0.15 +/- 0.05 (mean +/- SE, n = 6, P < 0.01), Cl absorption (pmol.mm-1 x min-1) from 30. 6 +/- 2.8 to 14.9 +/- 4.0 (P < 0.01), and potential difference (mV) from -5.4 +/- 1.3 to -4.0 +/- 1.2 (P < 0.01). Similar results were obtained in the lower concentration of 10(-10) M AVP and 10(-10) M ET-1. As for the osmotic water permeability (microns/s), 10(-8) M ET-1 significantly inhibited this from 320.1 +/- 50.9 to 202.1 +/- 42.2 (n = 7, P < 0.01) in the presence of 10(-9) M AVP.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of AVP and deoxycorticosterone on Na+ and water transport in the Dahl salt-sensitive rat CCD

Cortical collecting ducts (CCD) from inbred Dahl salt-sensitive rats were perfused in vitro to study effects of arginine vasopressin (AVP, present in the bath) and deoxycorticosterone pivalate (DOC) pretreatment on lumen-to-bath and bath-to-lumen fluxes of 22Na+ (J1----b and Jb----1 in pmol.min-1.mm-1, respectively), hydraulic conductivity (Pf, microns/s), and transepithelial voltage (VT, mV). J1----b was 37.1 +/- 5.3 (mean +/- SE) in untreated rats and increased to 83.2 +/- 15.9 with AVP. VT increased from -0.3 +/- 0.6 to -7.0 +/- 2.0. In DOC-pretreated rat CCDs, baseline J1----b was higher (85.1 +/- 7.6) as was VT (-11.3 +/- 1.7); J1b----b and VT were doubled with AVP addition (185.6 +/- 18.6 and -21.7 +/- 2.3, respectively). Thus J1----b in AVP-stimulated CCDs from untreated rats was not significantly different from control (no AVP) J1----b from DOC-pretreated rats; however, AVP produced a greater J1----b increase in the latter CCDs. Neither AVP nor DOC had an effect on Jb----1, which ranged from 25 to 50. Benzamil reduced J1----b to values not significantly different from Jb----1, and VT became zero in CCDs treated with both AVP and DOC, indicating Na+ transport stimulated by both hormones occurs through apical membrane Na+ channels. Pf increased from 59 +/- 80 to 1,072 +/- 176 with AVP addition to untreated rat CCDs and was unaltered by DOC or benzamil. Thus the Dahl rat CCD exhibits a transport response to AVP and DOC that is indistinguishable from that observed in Sprague-Dawley rats, as previously reported by this laboratory.

CAMP mediates the increase in apical membrane Na+ conductance produced in rat CCD by vasopressin

Experiments were conducted to determine if adenosine 3',5'-cyclic monophosphate (cAMP) mediates the stimulation of Na+ absorption by arginine vasopressin (AVP) in isolated perfused cortical collecting ducts (CCD) from rats treated with deoxycorticosterone pivalate (5 mg im) 5-9 days before study. AVP (220 pM) in the bathing solution hyperpolarized the transepithelial voltage (PDT) from -4.0 +/- 0.8 (SE) to -15.1 +/- 1.4 mV (n = 9, P less than 0.001) and decreased the transepithelial resistance (RT) from 40 +/- 8 to 33 +/- 6 omega.cm2 (n = 5, P less than 0.025). Bath addition of 0.2 mM dibutyryl cAMP (DBcAMP), 0.1 mM isobutylmethylxanthine (IBMX), 0.1 mM DBcAMP plus 0.1 mM IBMX, and 10 or 50 microM forskolin produced the same effects, reversibly hyperpolarizing PDT by 7.0-11.5 mV and decreasing RT by 6-12 omega.cm2. Addition of 10 microM amiloride to the luminal perfusate reduced PDT from -0.9 to +2.0 mV and increased RT in the presence or absence of any of the test agents. Addition of DBcAMP + IBMX or 50 microM forskolin to the bathing solution also reversibly depolarized the basolateral membrane voltage of principal cells by 1-2 mV and decreased the apical membrane fractional resistance from 0.82-0.84 to 0.72-0.77. Both effects were reversed by addition of amiloride to the luminal perfusate. These results demonstrate that cAMP is the intracellular mediator of the increase in apical membrane Na+ conductance produced by AVP in the rat CCD.

Differences in synergistic actions of vasopressin and deoxycorticosterone in rat and rabbit CCD

We examined the time course of changes in osmotic water permeability (Pf, micron/s), lumen-to-bath 22Na+ flux (Jl----b, pmol.min-1.mm-1), and transepithelial voltage (VT, mV) in response to arginine vasopressin (AVP) in isolated perfused rat and rabbit cortical collecting ducts (CCD). CCDs were isolated from "normal" (untreated) animals and animals 4-14 days after an intramuscular depot injection of deoxycorticosterone pivalate (DOC; 35 mg/rabbit, 5 mg/rat). In the normal rat CCD, 220 pM AVP added to the bathing solution increased Pf from approximately 0 to 981 +/- 120, Jl----b from 24 +/- 5 to 62 +/- 9, and VT from -0.7 +/- 0.6 to -4.0 +/- 0.6 (mean +/- SE; P less than 0.05). The effect of AVP on Pf in DOC-treated rats was not significantly different, but the effects on Jl----b and VT were greater. Jl----b rose from a control value of 78 +/- 16 to 192 +/- 14 with AVP, and VT hyperpolarized from -4.2 +/- 1.6 to -12.2 +/- 0.4. All of these effects of AVP in the normal and DOC-treated rat CCD persisted for greater than or equal to 90-150 min. In DOC-treated rabbit tubules, AVP caused a stable increase in Pf from 9 +/- 6 to 800 +/- 199, which was significantly greater than in normal tubules (379 +/- 32). In the normal rabbit CCD we confirmed previous observations that AVP produces only a transient increase in Na+ absorption and VT; Jl----b increased from 76 +/- 4 to 96 +/- 7 during the period 15-25 min after AVP addition but subsequently fell to 80 +/- 6; VT transiently hyperpolarized from -55 +/- 7 to -64 +/- 8 at 3-8 min after AVP and then fell to -54 +/- 9. In DOC-treated rabbit CCDs, AVP produced no change whatsoever in Jl----b, which was not significantly different from the control value of 99 +/- 7, nor in VT, which was -68 +/- 8 mV. These results indicate fundamental differences in the response of the rat and rabbit CCD to AVP and the presence or absence of synergistic interactions with mineralocorticoids, which may relate to their differing response to other autacoids.

The antiglucocorticoid and antiprogestin steroid RU 486: its glucocorticoid agonist effect is inadequate to prevent adrenal insufficiency in primates.

The glucocorticoid receptor antagonist RU 486 has been used to treat the hypercortisolism of patients with nonpituitary Cushing's syndrome. Since endogenous cortisol production fluctuates in many patients with either the ectopic ACTH syndrome or adrenocortical tumors, treatment of these patients with a fixed dose of RU 486 introduces the risk of adrenal insufficiency. While RU 486 possesses some glucocorticoid agonist activity in addition to its potent antagonist effects, it is not known whether this intrinsic agonist activity is of sufficient magnitude to prevent adrenal insufficiency and sustain life. To answer this question three groups of bilaterally adrenalectomized cynomolgus monkeys (n = 5/group) were randomized to receive a daily injection of RU 486 (5 mg/kg.day), cortisol (1.25 mg/kg.day), or saline (placebo). All adrenalectomized monkeys received weekly im injections of deoxycorticosterone pivalate (1 mg) to prevent mineralocorticoid deficiency. Five sham-adrenalectomized monkeys served as controls and received im injections of saline (placebo). Blood was collected before adrenalectomy or sham operation and every 3 days postoperatively for measurement of serum electrolytes, blood urea nitrogen, and creatinine; plasma ACTH concentrations; and complete blood and differential cell counts. All sham-operated and cortisol-replaced adrenalectomized monkeys survived, and none developed overt biochemical evidence of adrenal insufficiency. All placebo and RU 486-replaced adrenalectomized monkeys expired within 33 days after adrenalectomy, presumably from adrenal insufficiency. These findings suggest that while RU 486 is a partial glucocorticoid agonist, its degree of glucocorticoid agonism is inadequate to prevent adrenal insufficiency and support life in adrenalectomized primates.

Area postrema ablation and vascular reactivity in deoxycorticosterone-salt-treated rats.

In rats, central administration of the neurotoxin 6-hydroxydopamine prevents hypertension and certain functional vascular changes after deoxycorticosterone (DOC)-salt treatment. In this study, the effect of electrolytic ablation of the area postrema on blood pressure and vascular reactivity in DOC-salt-treated rats was examined. Four treatment groups of rats were studied (n = 5 in each): area postrema lesion, DOC-salt (DOC pivalate, 5 mg/wk s.c. for 5 weeks); sham lesion, DOC-salt; area postrema lesion, control; and sham lesion, control. Helically cut strips of carotid artery, aorta, and mesenteric artery were prepared for isometric force recording. Area postrema lesion attenuated hypertension in DOC-salt rats (mean arterial pressure, 107 vs 123 mm Hg in area postrema lesion and sham lesion rats, respectively; chronic aortic catheter). Vascular strips from sham lesion-control rats. These changes in vascular reactivity also were observed in area postrema lesion-DOC-salt rats. DOC treatment in rats on a normal sodium intake did not result in hypertension or increased vascular reactivity. In summary, integrity of the area postrema is necessary for hypertension but not for changes in vascular reactivity, in DOC-salt rats. It appears that 1) changes in vascular reactivity may be necessary, but they are not sufficient to produce DOC-salt hypertension, and 2) if these vascular changes are secondary to a central nervous system effect, they are mediated by a pathway distinct from the area postrema.

Opiate receptors and the endorphin-mediated cardiovascular effects of clonidine in rats: evidence for hypertension-induced mu-subtype to delta-subtype changes.

Effects of opiate receptor antagonists on centrally mediated cardiovascular responses to clonidine and beta-endorphin were studied in urethane-anesthetized spontaneously hypertensive Okamoto-Aoki rats (SHR), normotensive Sprague-Dawley rats, and Sprague-Dawley rats made hypertensive with deoxycorticosterone pivalate/salt. Microinjection of 270 pmol of naloxone into the nucleus tractus solitarii (NTS) significantly inhibited the hypotensive and bradycardic response to 5 nmol of similarly administered clonidine in both SHR and normotensive Sprague-Dawley rats. In SHR, a similar inhibition was observed after the delta-opiate receptor antagonist ICI 174864, but not after the mu-receptor antagonist beta-funaltrexamine (both at 270 pmol, intra-NTS), whereas in normotensive Sprague-Dawley rats, beta-funaltrexamine, but not ICI 174864, was an effective inhibitor. The same pattern of differential inhibition was seen when clonidine was given i.v. and the opiate antagonists were given intracisternally in SHR and Sprague-Dawley rats. Intra-NTS microinjection of 280 fmol of beta-endorphin caused hypotension and bradycardia, and these effects were similarly inhibited by ICI 174864 in SHR and by beta-funaltrexamine in Sprague-Dawley rats. In Sprague-Dawley rats made hypertensive by chronic administration of deoxycorticosterone pivalate and salt, the hypotensive and bradycardic effects of intra-NTS clonidine were inhibited by ICI 174864, but not by beta-funaltrexamine, a pattern similar to that in SHR, but different from that in normotensive Sprague-Dawley rats. These results support the hypothesis that beta-endorphin release and subsequent stimulation of opiate receptors in the NTS are involved in the cardiovascular effects of clonidine in rats. These results further suggest, however, that hypertension regulates the subtype of opiate receptors mediating these effects.