Functional characterization of the alpha adrenergic receptor modulating the hydroosmotic effect of vasopressin on the rabbit cortical collecting tubule.

To characterize the type of alpha adrenergic receptor, the effects of specific alpha adrenergic agonists and antagonists on antidiuretic hormone [( Arg8]-vasopressin [AVP])-induced water absorption were evaluated in cortical collecting tubules isolated from the rabbit kidney and perfused in vitro. In the presence of AVP (100 microU/ml), net fluid volume absorption (Jv, nanoliters per minute per millimeter) was 1.39 +/- 0.09 and osmotic water permeability coefficient (Pf, X 10(-4) centimeters per second) was 150.2 +/- 15.0. The addition of 10(-6) M phenylephrine (PE), an alpha adrenergic agonist, resulted in a significant decrease in Jv and Pf to 0.72 +/- 0.11 (P less than 0.005) and 69.9 +/- 10.9 (P less than 0.005). The addition of 10(-4) M prazosin (PZ), an alpha adrenergic antagonist, did not cause any significant change in Jv and Pf, which were 0.71 +/- 0.09 (P = NS vs. AVP + PE) and 67.8 +/- 9.5 (P = NS vs. AVP + PE), respectively. In a separate group of tubules, in the presence of AVP (100 microU/ml) and PE (10(-6) M), Jv and Pf were 0.78 +/- 0.17 and 76.1 +/- 18.0, respectively. The addition of 10(-6) M yohimbine (Y), an alpha 2 adrenergic antagonist, resulted in a significant increase in Jv to 1.46 +/- 0.14 (P less than 0.01) and Pf to 157.5 +/- 22.3 (P less than 0.005). Y (10(-4) M) or PZ (10(-4) M) alone did not significantly affect Jv and Pf in the presence of AVP )100 microU/ml). The effect of the natural endogenous catecholamine norepinephrine (NE) on Jv and Pf in the presence of AVP and propranolol (PR) was next examined. Jv and Pf were 1.53 +/- 0.07 and 176.3 +/- 5.2, respectively, in the presence of AVP (100 microU/ml) and PR (10(-4) M). The addition of NE (10(-8) M) resulted in a significant decrease in Jv to 1.19 +/- 0.11 (P less than 0.05) and Pf to 127.0 +/- 11.3 (P less than 0.02). Increasing the concentration of NE to 10(-6) M resulted in a further decrease in Jv and Pf to 0.70 +/- 0.10 (P less than 0.01 vs. NE 10(-8) M) and 68.5 +/- 10.6 (P less than 0.01 vs. NE 10(-8) M), respectively. The inhibitory effect of NE on AVP-induced water absorption was blocked by Y, but not by PZ. The effect of the alpha 2 adrenergic agonist clonidine (CD) on Jv and Pf was also examined. In the presence of AVP (10 microU/ml) Jv and Pf were 1.65 +/- 0.04 and 175.1 +/- 13.1, respectively. The addition of CD (10(-6) M) resulted in a significant decrease in Jv to 1.08 +/- 0.12 (P < 0.01) and Pf to 108.1 +/- 15.4 (P < 0.01). Increasing the concentration of CD to 10(-4) M resulted in a further significant decrease in Jv and Pf to 0.57 +/- 0.13 (P < 0.02 vs. CD 10(-6) M) and 54.7 +/- 13.8 (P < 0.01 vs. CD 10(-6) M), respectively. Similar results were obtained in the presence of AVP (100 microU/ml). The inhibitory effect of CD on AVP-induced water absorption was blocked by Y. CD did not significantly affect Jv and Pf in the presence of 8-bromo adenosine 3',5'-cyclic monophosphate. These studies indicate that alpha adrenergic agonists directly inhibit AVP-mediated water absorption at the level of renal tubule, an effect that can be blocked by specific alpha2 adrenergic antagonists, but not by specific alpha1 adrenergic antagonists. Alpha2 adrenergic stimulation directly inhibits AVP-mediate water absorption at the level of the tubule, an effect that can be blocked by a specific alpha2 adrenergic antagonist. This effect appears to be exerted at the level of activation of adenylate cyclase, since it is absent in the present of cyclic AMP.     

Endogenous Angiotensin Stimulation of Vasopressin in the Newborn Lamb

The effect of furosemide on plasma renin, vasopressin (AVP), and aldosterone concentrations was studied in 10 control and 6 nephrectomized lambs during the 1st 2 wk of life. In a separate study in 10 newborn lambs, 1-sarcosine-8-alanine-angiotensin II (saralasin acetate, 5 mug/kg per min) was infused alone for 40 min, after which furosemide 2 mg/kg i.v. was injected in association with continuing saralasin acetate infusion.Plasma renin activity increased from a mean (+/-SEM) of 21.3+/-3.4 ng/ml per h in the 10 control lambs to 39.4+/-8.2 ng/ml per h at 8 min (P < 0.001) and remained high through 120 min after furosemide. Plasma AVP and aldosterone concentrations increased from respective mean values of 2.1+/-0.4 muU/ml and 12.8+/-2.5 ng/dl to 9.8+/-2.0 muU/ml (P < 0.01) and 23.0+/-7.7 ng/dl (P < 0.05) at 35 min and 13.8+/-2.1 muU/ml and 23.0+/-4.4 ng/dl at 65 min after furosemide (each P < 0.01). There was an insignificant AVP response in the 10 lambs treated with angiotensin inhibitor: from a mean base line of 4.7+/-0.9 to 8.3+/-2.0 muU/ml at 35 min, and 7.4+/-2.0 muU/ml at 65 min after furosemide. There was no increase in AVP in the anephric lambs.The mean increment AVP response from base line in the newborn lambs without saralasin, Delta 10.8+/-2.0 muU/ml, was greater than in the lambs with saralasin, Delta4.0+/-1.9 (P < 0.05), and greater than in the anephric lambs, Delta3.3+/-2.1 muU/ml (P < 0.05). The mean blood pressure fell 6 mm Hg in the 10 control lambs (P < 0.05), 7 mm Hg in the anephric lambs (P < 0.05), and 16 mm Hg in the lambs treated with angiotensin inhibitor (P < 0.05) by 35 min after furosemide. However, the changes in plasma AVP were not related to the fall in blood pressure.These data support the view that the observed AVP response to furosemide in the newborn lamb was mediated through the renin-angiotensin system.     

Cytochrome P450 metabolites of arachidonic acid are potent inhibitors of vasopressin action on rabbit cortical collecting duct.

AA is metabolized by a cytochrome P450, NADPH-dependent epoxygenase to four regioisomeric epoxyeicosatrienoic acids (EETs). The EETs are further hydrated enzymatically to their respective diols, vic-dihydroxyeicosatrienoic acids (DHETs). We studied the effect of pretreatment with DHETs on 10 microU/cm2 arginine vasopressin (AVP)-stimulated hydraulic conductivity (Lp) (Lp x 10(-7) cm/atm/s, mean +/- SE) in rabbit cortical collecting ducts (CCDs) perfused in vitro at 37 degrees C. At 10(-6) M all four DHETs were potent inhibitors of the hydroosmotic effect of AVP. 14,15-DHET was the most potent isomer; it reduced AVP-induced Lp from a control value of 234.75 +/- 11.7, n = 17, to a value of 95.2 +/- 8.39, n = 5, P less than 0.0001, a reduction of AVP-mediated water flow of 60%. The inhibitory effect of 14,15-DHET was dose dependent and significant to nanomolar concentrations. 14,15-DHET at 10(-7) M was as potent an inhibitor of AVP's activity as was 10(-7) M PGE2. AVP's hydroosmotic effect is mediated through its intracellular second messenger, cAMP. 8-p-Chlorophenylthio-cAMP (CcAMP) at 10(-4) M induced a peak Lp of 189.6 +/- 11.0, n = 8; pretreatment with 10(-6) M 14,15-DHET reduced CcAMP-peak Lp to 132.0 +/- 13.4, n = 5, P less than 0.01, demonstrating a post-cAMP effect. Gas chromatography/mass spectroscopy suggests that EETs are present in extracts purified from CCDs. We conclude that cytochrome P450 epoxygenase eicosanoids are potent inhibitors of the hydroosmotic effect of vasopressin and are endogenous constituents of normal CCDs, the major target tissue for AVP.     

Phorbol myristate acetate, dioctanoylglycerol, and phosphatidic acid inhibit the hydroosmotic effect of vasopressin on rabbit cortical collecting tubule.

We explored the role for protein kinase C (PKC) in modulating vasopressin (AVP)-stimulated hydraulic conductivity (Lp) in rabbit cortical collecting tubule (CCT) perfused in vitro at 37 degrees C. In control studies, 10 microU/ml AVP increased Lp (mean +/- SE, X 10(-7) centimeters/atmosphere per second) from 4.4 +/- 0.9 to 166.0 +/- 10.4. Pretreatment with dioctanoylglycerol (DiC8) suppressed AVP stimulated peak Lp (peak Lp, 21.9 +/- 3.1). Pretreatment with 10(-9) and 10(-7) M 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) also blocked the increase in Lp in a dose-dependent fashion (peak Lp, 59.3 +/- 7.5 and 18.6 +/- 4.8, respectively). Inactive phorbol ester, 4 alpha-phorbol 12 beta,13 alpha-didecanoate (10(-7) M), had no effect. PMA also suppressed the increase in Lp induced by 10(-4) M 8-p-chlorophenylthio-cyclic AMP (CcAMP): peak Lp was 169.4 +/- 14.9 in control, 79.2 +/- 5.5 with 10(-9) M PMA, and 25.7 +/- 2.9 with 10(-7) M PMA. Furthermore, when 10(-7) M PMA was added to the bath 10 min after exposure to AVP, the Lp response to AVP was blocked. Peak Lp was 52.4 +/- 9.6 with PMA vs. 165.1 +/- 10.0 in control. Phosphatidic acid (PA), which is thought to stimulate phosphatidylinositol (PI) turnover, produced similar inhibitory effects on AVP as well as CcAMP-stimulated Lp: PA suppressed 10-microU/ml AVP-induced peak Lp from a control value of 159.6 +/- 7.9 to 88.9 +/- 15.8, and 10(-4) M CcAMP induced peak Lp from 169.4 +/- 14.9 to 95.5 +/- 7.7. We conclude that PMA, at concentrations known to specifically activate PKC, suppresses the hydroosmotic effect of AVP on CCT; This suppression is primarily a post-cAMP event; Inhibition of AVP-stimulated Lp by DiC8 and PA also suggests an inhibitory role for the PKC system; The ability of pre- and post-AVP administration of PMA to blunt the AVP response suggests that agents that act through modulation of PI turnover in CCT may regulate the hydroosmotic effect of AVP.     

Effects of fentanyl on vasopressin secretion in human subjects

To test the influence of an opioid on vasopressin (AVP) secretion, plasma AVP concentration was measured in five semirecumbent unmedicated volunteers before and during two continuous i.v. infusions of fentanyl. Infusion rates were adjusted to produce steady-state plasma fentanyl concentrations of 2.0 +/- 0.4 and 4.1 +/- 0.6 ng/ml; mild to moderate hypercarbia was induced during the control and infusion periods. Fentanyl increased plasma AVP concentration in a dose-dependent manner to 559 +/- 215 and 929 +/- 199% of the basal level of 1.9 +/- 0.7 pg/ml. Neither mild hypercarbia (PCO2 = 50 +/- 1 mm Hg) in the absence of fentanyl nor moderate hypercarbia (PCO2 = 66 +/- 3 mm Hg) in the presence of fentanyl changed plasma AVP concentration. Neither fentanyl nor hypercarbia, nor the combination of the two, altered plasma renin activity.     

Changes in the metabolic clearance of vasopressin and in plasma vasopressinase throughout human pregnancy.

Metabolic clearance rates (MCR) of arginine vasopressin (AVP) were measured serially in five women starting before conception, during gestational weeks 7-8 (early), 22-24 (middle), and 36-38 (late pregnancy), and again 10-12 wk postpartum. Hormonal disposal rates were determined after water loading to suppress endogenous AVP release using a constant infusion method designed to achieve three different steady-state concentrations of plasma AVP (PAVP) on each test occasion. Dose schedules were altered in mid- and late pregnancy to obtain comparable AVP levels at each stage of the protocol. Prehydration decreased plasma osmolality sufficiently to suppress AVP release, as circulating AVP-neurophysin measured serially in three of the women was undetectable. The MCR of AVP was similar before conception (0.75 +/- 0.31, 0.79 +/- 0.34, and 0.76 +/- 0.28 liters/min at PAVP of 2.6 +/- 1.9, 4.7 +/- 2.4, and 8.3 +/- 3.9 pg/ml), in early pregnancy (0.89 +/- 0.34, 0.97 +/- 0.04, and 0.95 +/- 0.40 liters/min at PAVP of 2.2 +/- 2.1, 3.9 +/- 3.2, and 7.9 +/- 3.4 pg/ml), and postpartum (0.70 +/- 0.21, 0.69 +/- 0.24, and 0.75 +/- 0.20 liters/min at PAVP 3.5 +/- 1.8, 5.1 +/- 3.7, and 9.1 +/- 4.2 pg/ml). Values at mid-pregnancy (2.8 +/- 1.3, 3.0 +/- 1.2, and 2.7 +/- 1.2 liters/min at PAVP 2.3 +/- 2.2, 4.0 +/- 3.6, and 7.7 +/- 3.9 pg/ml) and late pregnancy (3.2 +/- 1.4, 3.3 +/- 1.4, and 2.9 +/- 1.2 liters/min at PAVP 1.9 +/- 2.0, 3.8 +/- 2.6, and 7.4 +/- 4.1 pg/ml) increased 3-4-fold (all P less than 0.01). Plasma vasopressinase, undetectable at 7-8 gestational wk, increased markedly by mid- and slightly more by late gestation. Finally, relationships between PAVP and urine osmolality were similar before, during, and after pregnancy. We conclude that marked increments in the MCR of AVP occur between gestational weeks 7 and 8 and mid-pregnancy, which parallel the period of greatest rise in both trophoblastic mass and plasma vasopressinase. There was no evidence of a renal resistance to AVP during gestation.     

Inhibition by lithium of the hydroosmotic action of vasopressin in the isolated perfused cortical collecting tubule of the rabbit

Because treatment with lithium salts may impair renal concentrating ability, we investigated the possibility of a direct effect of lithium ions on the permeability to water of the collecting duct epithelium. The coefficient of hydraulic conductivity (Lp) of isolated perfused rabbit cortical collecting tubules (CCT) was measured in the presence and absence of arginine-8-vasopressin (AVP), or 8-bromo (Br) cyclic AMP (cAMP) and/or lithium chloride (Li 10 mM). In the absence of AVP, Li in the lumen for 30 min failed to affect basal water permeability; however, in tubules preincubated with Li in the lumen for 80 min, basal water permeability was reduced to 30% of the value found in control tubules (P less than 0.01). In CCT incubated at 25 degrees C with Li in the lumen for 3 h, the hydroosmotic response to 2.5 microU X ml-1 AVP (Lp = 6.88 +/- 1.54 nl X cm-2 X s-1 X atm-1) was significantly lower than that in the control tubules (13.98 +/- 1.59, P less than 0.01); the inhibition was not reversible. When Li was present in the peritubular medium only, the hydroosmotic effect of AVP was not different from that of the controls. The hydroosmotic effect of 25 microU/ml AVP was investigated at 37 degrees C. CCT exposed to Li in the lumen had a 49% inhibition of peak Lp under AVP (Lp = 10.98 +/- 1.17) as compared with control tubules (Lp = 21.39 +/- 1.51; P less than 0.005). In contrast, the hydroosmotic response to 8-Br-cAMP was not affected by lithium. The results are compatible with the view that Li inhibits the action of AVP at the level of the regulating protein or the catalytic unit of the membrane adenylate cyclase and that the site of the interaction can be reached by lithium only from the cytoplasmic side. The Li-antidiuretic hormone (ADH) interaction found here may represent the earliest pathophysiological event underlying the renal concentrating defect observed after Li administration.     

Effect of vasopressin on electrical potential difference and chloride transport in mouse medullary thick ascending limb of Henle''s loop.

Medullary thick ascending limbs of Henle's loop of the Swiss-Webster mouse were perfused in vitro with an isotonic perfusate and a Ringer's bathing medium. In five studies, addition of a supramaximal concentration of synthetic arginine vasopressin (AVP) to the bathing medium resulted in an increase in electrical potential difference (PD) from 5.0 +/- 1.5 mV, lumen positive, to 10.7 +/- 1.4 mV (P < 0.001). When AVP was removed, the PD returned to 2.6 +/- 0.9 mV (P < 0.001), then increased again to 6.9 +/- 1.7 mV (P < 0.01) when AVP was added a second time. A significant, but submaximal, increase in PD of 2.3 +/- 0.6 MV (P < 0.05) was observed in five medullary thick ascending limbs when AVP was added to the bathing medium at a concentration of 10 microunits/ml. This increase was approximately one-third of the response observed at a concentration of 100 microunits/ml in the same tubule. No further increment in PD was observed in five medullary thick ascending limbs when the AVP concentration was increased from 100 to 1,000 microunits/ml. In seven thick ascendcing limbs, the effect of AVP on PD was reproduced by the addition of 8-[p-chlorophenylthio]-cyclic 3',5'-adenosine monophosphate to the bathing medium at a final concentration of 0.1 mM. AVP increased unidirectional chloride flux from lumen to bath from 29.3 +/- 3.2 to 69.8 +/- 6.2 peq/cm per s (P < 0.001) in spite of an increase in the lumen positive PD from 1.6 +/- 0.5 mV to 7.0 +/- 0.6 mV (P < 0.001). Unidirectional chloride flux from bath to lumen was not affected by AVP. In another series of experiments, net chloride flux increased from 15.6 +/- 3.0 to 41.7 +/- 5.3 peq/cm per s (P < 0.05) after addition of AVP. The effect of AVP on hydraulic water permeability (Lp) was examined by adding raffinose to the bathing medium in both the presence and the absence of AVP. The calculated Lp of 16 +/- 2 nm/s per atm in the absence of AVP, although very low, was significantly different from zero (P < 0.01). However, the Lp did not increase significantly when AVP was added to the bathing medium. These results suggest that AVP has a second site of action in the kidney to increase chloride transport by the medullary thick ascending limb in addition to its well-known effect on the water permeability of the collecting tubule. The former effect would contribute to urinary concentrating ability by increasing the axial osmotic gradient in the renal medulla.     

Nifedipine attenuates both alpha-1 and alpha-2 adrenoceptor-mediated pressor and vasoconstrictor responses in conscious dogs and primates

Effects of the calcium channel blocker, nifedipine, were examined on the pressor and vasoconstrictor responses to stimulation of alpha-1 and alpha-2-adrenoceptors in chronically instrumented conscious dogs and Rhesus monkeys. Norepinephrine (NE), a mixed alpha-1 and alpha-2 adrenoceptor agonist, phenylephrine (PE) and methoxamine (M), selective alpha-1 adrenoceptor agonists, and B-HT 920, a selective alpha-2 adrenoceptor agonist, were injected i.v. after ganglionic (hexamethonium), beta adrenoceptor (propranolol) and muscarinic receptor (atropine methyl bromide) blockade. In the dog, NE (0.1 microgram/kg i.v.), PE (1 microgram/kg i.v.), M (20 micrograms/kg i.v.) and B-HT 920 (1 microgram/kg i.v.) produced similar increases in mean arterial pressure (NE, 52 +/- 4 mmHg; PE, 42 +/- 4 mm Hg; M, 43 +/- 7 mm Hg; B-HT 920, 45 +/- 6 mm Hg) and total peripheral resistance (NE, 20.8 +/- 5.8 mm Hg/l/min; PE, 23.1 +/- 4.2 mm Hg/l/min; M, 18.2 +/- 2.1 mm Hg/l/min; B-HT 920, 24.8 +/- 7.1 mm Hg/l/min). Nifedipine (0.5 microgram/kg/min i.v.) caused a similar attenuation of the pressor (NE, -54 +/- 8%; PE, -43 +/- 8%; M, -49 +/- 6%; B-HT 920, -56 +/- 8%) and vasoconstrictor (NE, -66 +/- 11%; PE, -52 +/- 9%; M, -60 +/- 13%; B-HT 920, -57 +/- 10%) responses to each of the alpha-adrenoceptor agonists. Nifedipine also attenuated pressor responses to alpha-1 and alpha-2 adrenoceptor agonists similarly in conscious monkeys. Thus, in conscious dogs and monkeys, calcium channel blockade attenuates similarly both alpha-1 and alpha-2 adrenoceptor-mediated vasoconstriction.     

Low density lipoprotein enhances the cellular action of arginine vasopressin in rat glomerular mesangial cells in culture.

The present study was undertaken to determine whether low density lipoprotein (LDL) modulates the cellular action of arginine vasopressin (AVP) in rat glomerular mesangial cells in culture. AVP increased cellular free calcium ([Ca2+]i) in a dose-dependent manner. When cells were preincubated for 24 h with 10 microgram/ml LDL, the 1 x 10(-7) M AVP-mobilized [Ca2+]i was 874 nM, a value significantly greater than that of 375 nM in the intact cells. AVP caused a biphasic change in cellular pH (pHi), namely, an early acidification followed by a sustained alkalinization, and the change in pHi produced by AVP was also enhanced by LDL. AVP stimulated a 2.2-fold increase in [3H]thymidine incorporation, an effect significantly greater in the presence of 10 micrograms/ml LDL. Furthermore, 1 x 10(-7) M AVP significantly activated mitogen-activated protein kinase from 14.0 to 24.5 pmol/mg protein. Such an activation was significantly enhanced by the LDL pretreatment. Both [3H]thymide incorporation and mitogen-activated protein kinase were not altered by 10 micrograms/ml LDL. [3H]AVP receptor binding was not affected by the LDL pretreatment. 1 x 10(-7) M AVP increased inositol trisphosphate production by 1.9-fold, an effect significantly greater in the presence of LDL. These results indicate that LDL enhances the cellular action of AVP and the AVP-stimulated cellular proliferation in glomerular mesangial cells. A site of action of LDL is the hydrolysis of phosphatidylinositol.     

Human vascular vasopressin receptors: analysis with selective vasopressin receptor antagonists

The vascular activity of arginine vasopressin (AVP) and selective AVP receptor antagonists was investigated in isolated arterial ring segments from human superior mesenteric arteries. AVP elicited a potent and concentration-dependent contraction in human mesenteric arterial rings with an EC50 value of 2.01 X 10(-9) M. The presence or absence of the vascular endothelium did not affect significantly AVP-induced contraction. AVP induced slight, although significant, tachyphylaxis in human mesenteric arteries. The selective vascular (V1) receptor antagonist [d(CH2)5 1Tyr(Me)2]AVP (SK&F 100273) shifted the concentration-response curves for AVP-induced vascular contraction to the right in a parallel manner (KB = 2.23 X 10(-9) M). A mixed V1/V2 receptor antagonist, [d(CH2)5 1D-Tyr(Et)2Val4desGly9]AVP (SK&F 101926), was also a potent antagonist of AVP-mediated vascular contraction; however, inhibition was marked by a nonparallel shift of the concentration-response curves with depression of maximum contraction. Furthermore, a relatively renal (V2) selective receptor antagonist [d(CH2)5 1D-Ile2Val4]AVP (SK&F 101485) was approximately 100-fold less potent at inhibiting AVP-induced vascular contraction (KB = 1.37 X 10(-7) M). These studies illustrate for the first time the in vitro effects of selective vasopressin receptor antagonists in isolated human blood vessels. Studies of other blood vessels and the design of therapeutically useful antagonists should proceed with the hypothesis that the vasopressin receptors mediating vascular contraction in human mesenteric arteries are of the V1 subtype.     

Human platelet fraction arginine-vasopressin. Potential physiological role.

Arginine-vasopressin (AVP) immunoreactivity (Ir) has been found to be elevated in platelet-rich plasma. PlatAVP was defined as platelet-rich plasma Ir minus platelet-poor plasma Ir (Pavp). PlatAVP, Pavp, and synthetic AVP were found to have identical retention time on high performance liquid chromatography analysis and similar mobility on thin-layer chromatography. During a standard osmotic suppression-stimulation test, Pavp increased with plasma osmolality (Posm, mosmol/kg H2O); Pavp (pg/ml) = 0.98 (Posm -274.4), r = 0.57, P less than 0.001, n = 65; but PlatAVP was not significantly correlated with Posm and remained at 5 pg/ml. This PlatAVP concentration was estimated to represent a true intraplatelet AVP concentration of 0.4 to 3.7 X 10(-9) M. Binding studies on intact human platelets demonstrated specific binding sites for [3H]AVP (n = 16; BMax = 98 +/- 30 binding sites/platelet; Kd = 0.72 +/- 0.24 nM). This in vitro affinity association constant (Kd) was close to the estimated in vivo intraplatelet AVP concentration. Measurement of PlatAVP could estimate vasopressin bound to a specific platelet receptor.     

Luminal vasopressin modulates transport in the rabbit cortical collecting duct.

We explored the action of luminal AVP in rabbit CCD perfused in vitro at 37 degrees C. Nanomolar concentrations of luminal AVP induced a sustained hyperpolarization of transepithelial voltage (Vt) in contrast to a transient hyperpolarization caused by basolateral AVP. 10 microM basolateral ouabain abolished the latter but not the former change in Vt. Despite a sustained hyperpolarization (from -20.7 +/- 2.9 to -34.1 +/- 4.7 mV; P less than 0.01), 10 nM luminal AVP only slightly altered net Na+ and K+ fluxes (7.6% stimulation and no significant change, respectively). Instead, luminal AVP appeared to modulate an acetazolamide-sensitive electrogenic ion transport because 200 microM basolateral acetazolamide suppressed the luminal AVP-induced hyperpolarization (percentage of Vt from -50.4 +/- 10.8 to -5.1 +/- 1.4; P less than 0.005). In terms of water transport, 10 nM luminal AVP did not change hydraulic conductivity (Lp, x 10(-7) cm/atm per s) (from 3.9 +/- 0.8 to 5.0 +/- 1.2), but suppressed the increase in Lp induced by 20 pM basolateral AVP (134.9 +/- 19.2 vs. 204.3 +/- 21.1 in control; P less than 0.05). These findings demonstrate distinct luminal action of AVP, suggesting amphilateral regulation of epithelial transport by AVP in the CCD.     

Effect of Intermittent Endogenous Hyperglucagonemia on Glucose Homeostasis in Normal and Diabetic Man

Infusion of glucagon causes only a transient increase in glucose production in normal and diabetic man. To assess the effect of intermittent endogenous hyperglucagonemia that might more closely reflect physiologic conditions, arginine (10 g over 30 min) was infused four times to 8 normal subjects and 13 insulin-dependent diabetic subjects (4 of whom were infused concomitantly with somatostatin to examine effects of arginine during prevention of hyperglucagonemia). Each arginine infusion was separated by 60 min. Diabetic subjects were infused throughout the experiments with insulin at rates (0.07-0.48 mU/kg per min) that had normalized base-line plasma glucose and rates of glucose appearance (Ra) and disappearance (Rd). Basal plasma glucagon and arginine-induced hyperglucagonemia were similar in both groups; basal serum insulin in the diabetics (16+/-1 muU/ml, P < 0.05) exceeded those of the normal subjects (10+/-1 muU/ml, P < 0.05) but did not increase with arginine. Serum insulin in normal subjects increased 15-20 muU/ml with each arginine infusion. In both groups each arginine infusion increased plasma glucose and Ra. Increments of Ra in the diabetics exceeded those of normal subjects, (P < 0.02); Rd was similar in both groups. In normal subjects, plasma glucose returned to basal levels after each arginine infusion, whereas in the diabetics hyperglycemia persisted reaching 151+/-15 mg/dl after the last arginine infusion. When glucagon responses were prevented by somatostatin, arginine infusions did not alter plasma glucose or Ra.CONCLUSIONS: Infusion of arginine acutely increases plasma glucose and glucose production in man solely by stimulating glucagon secretion; physiologic increments in plasma glucagon (100-150 pg/ml) can result in sustained hyperglycemia when pancreatic beta cell function is limited.     

The Physiological Role of Thyrotropin-Releasing Hormone in the Regulation of Thyroid-Stimulating Hormone and Prolactin Secretion in the Rat

The physiological role of thyrotropin-releasing hormone (TRH) in the regulation of thyrotropin (thyroid-stimulating hormone, TSH) and prolactin (Prl) secretion has been assumed but not proven. Stimulation of their release requires pharmacologic doses of TRH. Lesions of the hypothalamus usually induce an inhibition of TSH secretion and an increase in Prl. To determine whether TRH is essential for TSH and Prl secretion in the rat, 0.1 ml of TRH antiserum (TRH-Ab) or normal rabbit serum was administered to normal, thyroidectomized, cold-exposed, and proestrus rats through indwelling atrial catheter. Serum samples were obtained before and at frequent intervals thereafter. Serum TSH concentrations in normal, thyroidectomized, cold-exposed, and proestrus rats were not depressed in specimens obtained up to 24 h after injection of normal rabbit serum. In contrast, serum TSH was significantly decreased after the administration of TRH-Ab in all normal (basal, 41+/-8 muU/ml [mean+/-SE]; 30 min, 6+/-2; 45 min, 8+/-3; 75 min, 4+/-2); thyroidectomized (basal, 642+/-32 muU/ml; 30 min, 418+/-32; 60 min, 426+/-36; 120 min, 516+/-146); coldstressed (basal, 68+/-19 muU/ml; 30 min, 4+/-3; 180 min, 16+/-8); and proestrus (basal, 11 a.m., 57+/-10 muU/ml; 1 p.m., 20+/-3; 3 p.m., 13+/-4; 5 p.m., 19+/-3) rats. However, 0.1 ml of TRH-Ab had no effect on basal Prl concentrations in normal or thyroidectomized rats and did not prevent the Prl rise in rats exposed to cold (basal, 68+/-7 ng/ml; 15 min, 387+/-121; 30 min, 212+/-132; 60 min, 154+/-114), or the Prl surge observed on the afternoon of proestrus (basal 11 a.m., 23+/-2 ng/ml; 1 p.m., 189+/-55; 3 p.m., 1,490+/-260; 5 p.m., 1,570+/-286). These studies demonstrate that TRH is required for TSH secretion in the normal, cold-exposed and proestrus rat and contributes, at least in part, to TSH secretion in the hypothyroid rat, but is not required for Prl secretion in these states.     

Importance of Hepatic Portal Circulation for Insulin Action in Streptozotocin-Diabetic Rats Transplanted with Fetal Pancreases

The importance of the hepatic portal circulation in the response to insulin was assessed in streptozotocin-diabetic rats transplanted with syngeneic fetal pancreases. Partial reversal of diabetes was accomplished by transplantation of two or three fetal pancreases beneath the capsule of the kidney; complete reversal followed shunting of the venous drainage from the transplants to the liver. Plasma glucose after streptozotocin of 509+/-31 mg/dl (mean+/-SEM) fell after transplantation to 395+/-23 and after the shunt to 143+/-5 mg/dl. Urine volume fell from 84+/-4 to 50+/-5 ml/d and then to normal (17+/-1 ml/d) after the shunt. Glucose excretion which was 8.1+/-0.3 g/d after streptozotocin fell after transplantation to 4.8+/-0.3 g/d and after the shunt completely disappeared from the urine. The disappearance rate of glucose injected into the circulation, which was 0.50+/-0.07%/min in untreated diabetes, increased to 1.39+/-0.38%/min after transplantation and to 2.52+/-0.31%/min after the shunt, not different from normal controls (2.79+/-0.25). Plasma immunoreactive insulin (IRI) was below normal (25-35 muU/ml) and unresponsive to glucose in untreated diabetic rats. After transplantation IRI levels ranged from 73-223 muU/ml and there was no rise after glucose injection. After the shunt both the basal IRI (36+/-5 muU/ml) and the peak response to glucose at 10 min (58+/-7 muU/ml) were the same as in normal controls (42+/-4 and 62+/-7 muU/ml, respectively). The fall in IRI after the shunt is explained by increased extraction of insulin passing into the liver and also diminished secretion. After removal of the transplants plasma glucose and urine values returned almost to pretransplant levels.Secretion of insulin by transplanted pancreases into the liver enhances the effectiveness probably by increased extraction and action and reveals the importance of the normal route for insulin delivery.     

Dopamine during α- or β-Adrenergic Blockade in Man: HORMONAL, METABOLIC, AND CARDIOVASCULAR EFFECTS

We studied the contribution of alpha- and beta-adrenergic receptor activation to the cardiovascular, metabolic, and hormonal effects of dopamine. At a concentration of 1.5 mug/kg.min, the infusion of dopamine in 12 normal volunteers was associated with a transient but significant rise in pulse rate, which was prevented by propranolol. Venous plasma glucose did not change throughout the experiments, and a mild increase in plasma free fatty acid levels observed during the administration of dopamine alone was antagonized by propranolol. In contrast, neither the beta-adrenergic blocker, propranolol, nor the alpha-adrenergic blocker, phentolamine, was effective in inhibiting the dopamine-induced rise in plasma glucagon (from 82+/-9 to 128+/-14 pg/ml; P < 0.005) and serum insulin (from 7.5+/-1 to 13+/-1.5 muU/ml; P < 0.005) or its suppression of plasma prolactin (from 8.5+/-1 to 5.2+/-0.8 ng/ml; P < 0.001). Although serum growth hormone levels did not change during the infusion of dopamine alone, an obvious rise occurred in three subjects during the combined infusion of propranolol and dopamine.Whereas some metabolic and cardiovascular effects of dopamine are mediated through adrenergic mechanisms, these observations indicate that this is not the case for the effects of this catecholamine on glucagon, insulin, and prolactin secretion, and thus provide further support for the theory of a specific dopaminergic sensitivity of these hormonal systems in man.     

Effects of intravenous arginine vasopressin on epicardial coronary artery cross sectional area in a swine resuscitation model

Although arginine vasopressin (AVP) has been shown to be a promising drug during cardiopulmonary resuscitation (CPR), concern has been raised about the potential for AVP-mediated vasoconstriction of the coronary arteries. In a prospective, randomized laboratory investigation employing an established porcine model, the effects of AVP on haemodynamic variables, left anterior descending (LAD) coronary artery cross sectional area employing intravascular ultrasound (IVUS), and return of spontaneous circulation were studied. During sinus rhythm, the LAD coronary artery cross sectional area was measured by IVUS at baseline, and 90 s and 5 min after AVP (0.4 U/kg IV). Following a 60 min recovery, ventricular fibrillation was induced. At 4 min, chest compressions were initiated; AVP (0.4 U/kg IV) was injected at 5.5 min, and defibrillation performed at 8 min. LAD coronary artery cross sectional area was measured by IVUS at the pre-arrest baseline, 90 s after drug injection during CPR, and 5 min after return of spontaneous circulation. Compared with baseline, the mid-LAD coronary artery cross sectional area increased significantly (P<.05) 90 s and 5 min after AVP administration (9.2+/-.5mm2 versus 10.7+/-.6mm2 versus 11.7+/-.6mm2, respectively) during normal sinus rhythm. Similarly during ventricular fibrillation and CPR plus AVP, the mid-LAD coronary artery cross sectional area increased at 90 s after AVP compared with baseline (9.5+/-.6mm2 versus 11.0+/-.7mm2; P<.05). Moreover, the cross sectional area increased further 5 min after return of spontaneous circulation (9.5+/-.6mm2 versus 14.0+/-.8mm2, P<.05). In conclusion, in this experimental model with normal coronary arteries, AVP resulted in significantly increased LAD coronary artery cross sectional area during normal sinus rhythm, during ventricular fibrillation with CPR, and after return of spontaneous circulation.     

Effect of amiodarone on serum triiodothyronine, reverse triiodothyronine, thyroxin, and thyrotropin. A drug influencing peripheral metabolism of thyroid hormones.

2-n-Butyl-3-(4'-diethylaminoethoxy-3',5'-diiodobenzoyl)-benzofurane (amiodarone), a drug used in arrythmias and angina pectoris, contains 75 mg of organic iodine/200 mg active substance. Four studies were performed to test its effect on thyroid hormone metabolism: (a) nine male subjects were treated with 400 mg of amiodarone for 28 days; (b) five male subjects received, for the same period of time, 150 mg of iodine in the form of Lugol's solution; (c) five subjects received 300 mug L-thyroxine (T4) for 16 days; from the 10th to the 16th day, 400 mg of amiodarone was added; and (d) five euthyroid subjects received 300 mug L-T4 for 16 days. The changes in serum thyroid-stimulating hormone (TSH), serum total T4, 3,5,3'-triiodothyronine (T3), free T3, and 3,5',3'-triiodothyronine (reverse T3, rT3) were measured, and the pituitary reserve in TSH was evaluated by a thyrotropin-releasing hormone (TRH) test. The results show that amiodarone induced a decrease in serum T3 (28+/-5.1 ng/100 ml, mean+/-SEM, P less than 0.0S and 82.7+/-9.3 ng rT3/100 ml, P less than 0.01). The control study with an equal amount of inorganic iodine did not induce these opposite changes but slightly lowered serum rT3, T3, and T4. In the third study, serum rT3 increased as under amiodarone treatment, thereby proving that these changes were peripheral. It is suggested that amiodarone changes thyroid hormone metabolism, possibly by reducing deiodination of T4 to T3 and inducing a preferential production of rT3. Amiodarone also increased the response of TSH to TRH. The maximal increment of serum TSH above base line was 32+/-4.5 muU/ml under treatment and 20+/-3 muU/ml before treatment (P less than 0.01). During this test, the serum T3 increase was more pronounced than during the control period (83+/-13 and 47+/-7.4 ng/100 ml, P less than 0.05).     

Hyperresponse to Thyrotropin-Releasing Hormone Accompanying Small Decreases in Serum Thyroid Hormone Concentrations

To determine whether pituitary thyrotropin (TSH) responsiveness to thyrotropin-releasing hormone (TRH) is enhanced by small decreases in serum thyroxine (T4) and triiodothyronine (T3), 12 euthyroid volunteers were given 190 mg iodide po daily for 10 days to inhibit T4 and T3 release from the thyroid. Basal serum T4, T3, and TSH concentrations and the serum T4 and TSH responses to 400 mug TRH i.v. were assessed before and at the end of iodide administration. Iodide induced small but highly significant decreases in basal serum T4 (8.0+/-1.6 vs. 6.6+/-1.7 mug/100 ml; mean +/- SD) and T3 (128+/-15 vs. 110+/-22 ng/100 ml) and increases in basal serum TSH (1.3+/-0.9 vs. 2.1+/-1.0 muU/ml). During iodide administration, the TSH response to TRH was significantly increased at each of seven time points up to 120 min. The maximum increment in serum TSH after TRH increased from a control mean of 8.8+/-4.1 to a mean of 13.0+/-2.8 muU/ml during iodide administration. As evidence of the inhibitory effect of iodide on hormonal release, the increment in serum T3 at 120 min after TRH was significantly lessened during iodide administration (61+/-42 vs. 33+/-24 ng/100 ml). These findings demonstrate that small acute decreases in serum T4 and T3 concentrations, resulting in values well within the normal range, are associated both with slight increases in basal TSH concentrations and pronounced increases in the TSH response to TRH. These results demonstrate that a marked sensitivity of TSH secretion and responsiveness to TRH is applicable to decreasing, as well as increasing, concentrations of thyroid hormones.