Calcitonin gene-related peptides: their binding sites and receptor accessory proteins in adult mammalian skeletal muscles.

This work addresses the presence, pharmacological properties, and anatomical localization of calcitonin gene-related peptide-alpha (CGRPalpha) binding sites and the receptor's accessory proteins in endplate-enriched and non-endplate muscle membrane samples from adult rat gracilis muscles. We examined the binding of (125)I-[Tyr(0)]-CGRPalpha, the competitive binding of CGRPalpha analogs, the immunohistochemical localization of the receptor's accessory proteins, and Western blots of the receptor component protein. Results show that: (a). (125)I-[Tyr(0)]-CGRPalpha binding is saturable, specific, and consistent with the presence of a homogeneous population of binding sites (Hill coefficients=1.0) in endplate and non-endplate samples exhibiting dissociation constants of 0.39 nM and 0.38 nM, respectively; (b). the density of binding sites in the endplate samples (71.0 fmoles/mg protein) is considerably higher than that in their non-endplate counterparts (34.6 fmoles/mg protein); (c). unlabeled CGRPalpha, hCGRP8-37 and calcitonin compete with the radioligand with the same order of potency in the endplate and non-endplate samples; and (d). the localization of the receptor accessory proteins, including the receptor activity-modifying protein (RAMP1) and the receptor component protein (RCP), for the most part matches that of the motor end-plates. Thus, gracilis muscles express CGRPalpha-specific binding sites which are predominantly localized in the muscle's motor endplate regions where RAMP1, RCP, CGRPalpha, acetylcholine receptors, and acetylcholinesterase are detected in high concentrations. These findings imply that the CGRPalpha binding sites reflect the presence of physiologically functional receptors with a pharmacological profile consistent with that of the CGRPalpha receptor type 1 (CGRP1). When considered together with earlier studies on the same neuromuscular preparation, the present work further suggests that the motoneuron-dependent trophic control of acetylcholine receptors and acetylcholinesterase in skeletal muscle endplates is partly mediated by nerve-derived CGRPalpha activating specific receptors which are highly sensitive to the truncated peptide hCGRP8-37.

Chlorpropamide upregulates antidiuretic hormone receptors and unmasks constitutive receptor signaling.

The mechanism by which chlorpropamide (CP) treatment promotes antidiuresis is unknown. CP competitively inhibited antidiuretic hormone (ADH) binding and adenylyl cyclase (AC) stimulation (inhibition constants K(i) and K'(i) of 2.8 mM and 250 microM, respectively) in the LLC-PK(1) cell line. CP (333 microM) increased the apparent K(a) of ADH for AC activation (0.31 vs. 0.08 nM) without affecting a maximal response, suggesting competitive antagonism. Because CP lowers "basal" AC activity and the AC activation-ADH receptor occupancy relationship (A-O plots), it is an ADH inverse agonist. Twenty-four-hour CP exposure (100 microM) upregulated the ADH receptors without affecting affinity. This lowered K(a) and increased basal AC activity and maximal response (1. 86 vs. 1.35 and 14.9 vs. 10.6 fmol cAMP. min(-1). 10(3) cells(-1), n = 6, P<0.05). NaCl, which potentiates ADH stimulation, also increased basal AC activity. This, together with the CP-ADH inverse agonism and increased basal AC activity at higher receptor density, unmasks constitutive receptor signaling. The CP-ADH inverse agonism explains receptor upregulation and predicts the need for residual ADH with functional isoreceptors for CP-mediated antidiuresis. This could be why CP ameliorates partial central diabetes insipidus but not nephrogenic diabetes insipidus.

Diagnosis and management of diabetes insipidus during pregnancy.

OBJECTIVE: To provide an overview of diagnostic and treatment strategies in pregnant patients with diabetes insipidus (DI). METHODS: We review the changes in osmoregulation during normal pregnancy, characterize the various types of DI that can occur during pregnancy, and discuss the recommended management. RESULTS: The incidence of DI is 2 to 4 cases per 100,000 gestations. Central DI can precede pregnancy or manifest initially during gestation. With preexistent central DI, pregnancy usually aggravates the disorder, and the requirements for antidiuretic hormone (ADH) usually increase. Such an effect is less likely to be noted in ADH-independent nephrogenic forms of DI. Currently, the major type of DI associated with pregnancy is a transient syndrome that is resistant to arginine vasopressin (AVP) but responsive to desmopressin (dDAVP); such cases of DI are often associated with liver abnormalities or preeclampsia. This syndrome is explained by excess vasopressinase, a placental enzyme which degrades AVP but not dDAVP. A transient recurrent type of DI can occur during gestation in patients with limited ADH-secreting capacity and is responsive to both AVP and dDAVP. Latent central DI manifesting after complicated delivery and transient nephrogenic DI, resistant to both AVP and dDAVP, have also been reported. CONCLUSION: The differential diagnosis of polyuric and polydipsic states during pregnancy is broad, and precise diagnosis may be difficult. The use of dDAVP to treat DI during gestation has proved effective and safe for both the mother and the fetus.

Effect of iron on renal tubular epithelial cells.

Since iron has been implicated as a potential nephrotoxin, we examined the effect of iron on several aspects of cultured renal tubular epithelial cell biology. We found that exposure to 10(-4) M of either the ferrous or ferric form of iron impaired healing of denuded areas made within confluent monolayers of LLC-PK1 cells. This impairment required 30 to 80 hours of exposure to iron to occur and was also seen in another renal tubular epithelial cell line (MDCK cells). To delineate the potential mechanism(s) of this impairment, we examined the expression of a key integrin subunit involved in cell-matrix adhesion. Exposure of LLC-PK1 cells to 10(-4) M ferric citrate for 72 hours significantly decreased expression of the beta 1 integrin subunit as determined by flow cytometry. To determine if iron impairs another process that occurs at the basolateral surface, the effects of 72 hours of exposure to iron on adenylate cyclase activity were examined. Both ferric and ferrous citrate significantly enhanced vasopressin- and forskolin-stimulated adenylate cyclase activity. To examine if iron can regulate proliferation, the effect of iron on 3H-thymidine uptake was measured. We found that ferric citrate diminished proliferation and this decrease required the presence of either serum or transferrin. To ascertain if iron affected ultrastructure, we used transmission electron microscopy and found that iron accumulation within cells was much more apparent with ferric than ferrous citrate. Ferric iron induced mild-to-moderate cytopathic changes. These results indicate that iron is capable of inducing multiple changes in renal tubular epithelial function. The effect of iron to impair wound healing may be related to diminished expression of the beta 1 integrin subunit and perhaps to impaired proliferation.

Effect of bacitracin on the degradation of a vasopressin receptor ligand with high affinity for the V1 and V2 vasopressin isoreceptors.

We previously described a new iodinated vasopressin analogue (N epsilon-[125I]L-Tyr-[Lys8]-vasopressin) with high affinity for the vasopressin V1 and V2 isoreceptors. The aim of the present study was: i) to analyse the degradation pathway of N epsilon-[125I]L-Tyr-[Lys8]-vasopressin and (ii) to look for an effective inhibitor of radioligand degradation. N epsilon-[125I]L-Tyr-[Lys8]-vasopressin was processed in a temperature-dependent manner by crude cell membranes from LLC-PK1 cells. Only one degradation product was seen using RP-HPLC. The degradation product co-eluted with monoiodotyrosine. The stereoisomer, N epsilon-[125I]D-Tyr-[Lys8]-vasopressin, underwent the same degradation process. Bacitracin prevented degradation at doses as low as 40 mg/l without alterating the binding affinity.

Determination of specific activity of labeled ligands by nonlinear regression analysis.

The specific radioactivity (SA) of 125I-lysine vasopressin (LVP) was determined by analyzing the binding B (cpm/tube) of variable amounts of tracer T (cpm/tube) to a constant amount of an LVP antibody, in the presence of known quantities L (mol/tube) of LVP standards. The parameters of the equations B = f(F) and B/F = g(T), describing B as a function of free F (cpm/tube) tracer or the ratios B/F as a function of T, were first calculated by nonlinear regression analysis of the results obtained with tracer alone. Then the dependent variables B or B/F were measured in the presence of LVP and analyzed with the same equations by substituting the independent variables F or T with (F + alpha FL) and (T + alpha L), respectively, where alpha (cpm/mol) represents a measure of the SA and FL (FL = L.F/T), free LVP, respectively. The SA was thus treated as an unknown parameter to be calculated by nonlinear regression. This method was compared with the traditional interpolation of the SA from the self-displacement and standard curves. Tracer and ligand were found to have the same affinity for the binding sites, since the set of equations B = f(F + alpha FL) and B/F = g(T + alpha L), describing the binding of the tracer in the presence of LVP and equations B = f(F) and B/F = g(T) to which these equations reduce in the absence of LVP (L = 0), had identical binding parameters. To be valid, any method based on self-displacement requires that the tracer and standards have the same affinity for the binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

High specific activity 125I- and 35S-labeled vasopressin analogues with high affinity for the V1 and V2 vasopressin isoreceptors.

Since iodination of the tyrosine residue in the pressin ring of vasopressins abolishes binding to the V2 (renal) isoreceptor, the low specific activity tritiated vasopressins have been the only radioligands available for this receptor. Alternative vasopressin radioligands are described in the present study. N-tert-Butoxycarbonyl- (N-t-Boc) 125I-tyrosine or [35S]methionine were conjugated to the 8th amino acid of lysine- (LVP) or deamino-ornithine-vasopressin via active succinimidyl esters. Following the purification on C-18 reverse-phase high pressure liquid chromatography, t-Boc removal, and a second high pressure liquid chromatography purification, specific activities of 2200 and 1300 Ci/mmol were obtained for the 125I- and the 35S-labeled ligands, respectively. These vasopressin analogues, conjugated outside the pressin ring, were found to bind with high affinity to the V1A (vascular) and V2 vasopressin isoreceptors (Kd less than or equal to 10(-9) M) and to retain the full biological activity of intact vasopressin. The present study demonstrates the possibility of producing high specific activity radioligands with high affinity for the V1A and V2 vasopressin isoreceptors by conjugating labeled moieties to the 8th amino acid of vasopressin analogues. Since these new radioligands have specific activities much higher than the tritiated ligands (1300-2200 versus 10-30 Ci/mmol), they should provide considerable advantages in the future study of the physiology and biochemistry of the AVP receptors.

Internalization of V2-vasopressin receptors in LLC-PK1-cells: evidence for receptor-mediated endocytosis.

The mechanism of internalization of the vasopressin-receptor (V2-subtype) of LLC-PK1-cells, a pig renal tubular cell line, is unknown. We studied internalization utilizing a novel, highly specific vasopressin analogue ((125I)-[8-p(OH)-phenylpropionyl]-LVP, 2000 Ci/mmol). Scatchard analysis performed with membranes of LLC-PK1-cells revealed a Kd of 0.8 +/- 0.2 nM and a Bmax of 366 +/- 41 fmol/mg of protein. Degradation of the ligand was excluded by RP-HPLC-analysis. Internalization was proven by the acid-wash technique, quantitative light-microscopic autoradiography and electron microscopy. The ligand was internalized in a time- and temperature-dependent manner. At 4 degrees C, no uptake was found; at 22 degrees C, after 30 min of incubation, more than 50% of the radioligand was found inside the cell. Electron microscopy demonstrated that plasma-membrane bound vasopressin receptors are internalized by receptor-mediated endocytosis via coated pits.

Correlates of brain edema in uncontrolled IDDM.

Blood glucose, plasma sodium, bicarbonate (HCO3-), vasopressin, and hematocrit were monitored before and during treatment in patients with uncontrolled insulin-dependent diabetes mellitus (IDDM). These parameters were correlated with simultaneous serial cranial computed tomography readings of brain edema. Six of seven patients had positive computed tomography readings for brain edema on admission. Initial brain edema correlated directly with blood glucose (r = 0.79, P = 0.033) and inversely with HCO3- (r = -0.76, P = 0.047). At 6 h, brain edema still correlated with acidosis (HCO3-; r = -0.79, P = 0.033) but no longer with blood glucose. At that time, however, brain edema correlated with the rate of change in blood glucose (r = 0.915, P = 0.005). Results of interactive stepwise regression analysis suggest that the change in the calculated effective plasma osmolality plays a predominant role in the progression of brain edema during therapy (r = 0.995, P less than 0.001). Thus, although hyperglycemia and acidosis probably predispose to diabetic brain edema, osmotic factors may be major predictors of its evolution. No relationships were detected between brain edema and initiation of insulin therapy, plasma vasopressin, or changes in hematocrit. The factors responsible for initial brain edema and its progression, statistically identified in this study, require reassessment of common theories that attribute brain edema exclusively to therapy.

Aldosterone in congestive heart failure: analysis of determinants and role in sodium retention.

In patients with congestive heart failure (CHF), the role of aldosterone in the abnormal sodium (Na+) retention and the determinants of plasma aldosterone (PA) including plasma atrial natriuretic factor (hANF), plasma renin activity (PRA), and plasma potassium (K+) have not been fully elucidated. We therefore studied the effect of the specific aldosterone antagonist, spironolactone, on urinary Na+ and K+ excretion and plasma hormone responses in 6 Na(+)-retaining CHF patients. After withdrawal of diuretics 4 days prior to the study, the CHF patients were placed on a Na+ intake of 100 mmol/day for 9 days. Spironolactone, 200 mg p.o. bid, was administered for the last 4 days of the 9-day study period. PRA and norepinephrine increased with spironolactone treatment (both p less than 0.05). Plasma hANF before spironolactone was significantly elevated and decreased during spironolactone therapy (p less than 0.05). Urinary Na+ excretion significantly increased during spironolactone administration and the positive Na+ balance was reversed in the CHF patients. Moreover, the urine Na+:K+ concentration ratio significantly increased during spironolactone administration. Analysis of the relationship between PA, plasma K+, PRA, and plasma hANF indicated that PRA is the primary determinant of PA in patients with CHF. Thus, the present results indicate that the renin-angiotensin-aldosterone system is an important mediator of Na+ retention in CHF, as evidenced by the reversal of the positive Na+ balance with a specific aldosterone antagonist. This natriuretic effect can be demonstrated in the presence of potential antinatriuretic influences including stimulation of the renin-angiotensin and sympathetic nervous systems and a decrease in plasma hANF.

Osmoregulation of vasopressin in diabetic ketoacidosis.

Osmoregulation of arginine vasopressin (AVP) is altered in diabetic ketoacidosis (DKA). With hyperglycemia, the AVP-plasma sodium (PNa) curve is displaced to the left, whereas the AVP-osmolality (Posm) curve is displaced to the right. The shift in the Na curve is explained by either resetting of the Na set point or by glucose acting as a nonpermeable solute, substituting for Na. Conversely, putative unmeasured solutes that, like urea, fail to affect AVP have been postulated to account for the right shift in the AVP-Posm curve. Therefore the respective roles of Posm = sigma [Xi] and plasma tonicity (Pton = sigma [sigmaiXi]), i.e., the sum of concentrations of all solutes [Xi] corrected (Pton) or not (Posm) for their relative cell permeability (sigma i), were studied in DKA. Indeed, Posm = sigma [Xi] exceeds Pton = sigma [sigma iXi] in DKA, since sigma i less than 1 for glucose. Potential determinants of AVP release (Posm, Pton, and PNa) were monitored in 7 patients with DKA. Conventional correlation analysis and two-dimensional (2D) graphs reproduced the paradox of an opposite shift in PNa and Posm set points for AVP release. However, by using the concept of tonicity instead of osmolality, 3D plots instead of 2D graphs, and multiple regressions instead of correlations, the AVP-PNa and AVP-Pton curves did not appear displaced. The concept of tonicity resolved the paradox of both osmolality and Na thresholds reset in opposite directions. Indeed, in states where a solute like glucose (with sigma less than 1) contributes substantially to plasma osmolality, Posm measured in vitro by the osmometer greatly exceeds Pton perceived in vivo by the osmoreceptor.

Measurement of endogenous lithium levels in serum and urine by electrothermal atomic absorption spectrometry: a method with potential clinical applications.

A highly sensitive flameless atomic absorption method has been adapted for the determination of endogenous trace lithium levels in serum and urine. With ammonium nitrate as the only matrix modifier, serum levels of Li as low as 0.03 mumol/liter are measured accurately and there is no requirement for standard additions. The need for background correction during analysis was clearly established, and tungsten and Zeeman-effect background corrections were compared. The tungsten correction offered superior sensitivity and linearity of standards. Recoveries in urine and serum average 94.8 +/- 7.7 and 95.3 +/- 6.1% (+/- SD), respectively. The endogenous serum Li levels were 0.16 +/- 0.08 mumol/liter for normal subjects dwelling in the Denver metropolitan area. The mean 24-h excretion rate was 5.24 +/- 1.4 mumol/day. The mean fractional excretion of endogenous Li (clearance Li/clearance creatinine) was 23.2 +/- 3.0%, a value similar to values published for exogenously administered Li and measured by conventional methods.

Role of hemodynamic factors in osmoregulatory alterations of rat pregnancy.

Plasma osmolality (Posmol) decreases in pregnancy, possibly because of hemodynamically mediated arginine vasopressin (AVP) secretion, i.e., inadequate vascular filling and/or decreased blood pressure. This hypothesis was tested in Sprague-Dawley rats treated on gestational days 1-18 or 13-18 with 1) deoxycorticosterone acetate (DOCA) + standard chow (0.5% Na), 2) vehicle + standard chow, or 3) high-Na (1.25%) diet. Renal sodium "escape" and suppression of the renin-aldosterone system suggested "effective volume expansion," yet Posmol was similar in all pregnant groups, 7-10 mosmol/kg below levels in virgin controls, and plasma AVP was unaltered. Apparent osmotic thresholds for AVP secretion, similar in control and DOCA-treated gravid animals, were 8-10 mosmol/kg below those of untreated virgin rats. Norepinephrine + DOCA, administered to gravid animals consuming normal or high-Na chow, increased blood pressure approximately 10% above control levels, but this also failed to alter Posmol. These data suggest that mechanisms other than hemodynamically mediated AVP secretion are responsible for the osmoregulatory alterations accompanying rodent pregnancy.

Elevated plasma atrial natriuretic factor and vasopressin in high-altitude pulmonary edema.

A diagnosis of acute high-altitude pulmonary edema was made in five male skiers (age, 35.0 +/- 1.8 years) by history and physical examination and was confirmed by a characteristic chest radiogram showing alveolar infiltrates associated with a normal cardiac silhouette. Five healthy age- and sex-matched subjects with similar physical activity at the same altitude served as controls. Plasma sodium was 135.0 +/- 1.5 mmol/L in the acutely ill patients compared with 144.0 +/- 3.3 mmol/L in the controls (P less than 0.025). Mean plasma atrial natriuretic factor immunoreactivity averaged 17.6 +/- 5.6 pmol/L in patients with high-altitude pulmonary edema compared with 6.8 +/- 0.7 pmol/L in the controls at the same altitude (P less than 0.05). Elevated atrial natriuretic factor levels normalized to 7.5 +/- 1.9 pmol/L (P less than 0.05) during recovery in Denver (altitude, 1600 meters) 24 hours later. Plasma arginine vasopressin levels were 1.8 +/- 0.37 pmol/L in patients with high-altitude pulmonary edema at diagnosis compared with 0.92 +/- 0.28 pmol/L in controls (P = 0.07). The inappropriately elevated arginine vasopressin levels decreased to 1.29 +/- 0.37 pmol/L during recovery (P less than 0.025), but the lowered plasma sodium concentration had not normalized by discharge within 24-hours of transfer to Denver and averaged 135.8 +/- 1.2 mmol/L. The pathophysiologic implications of these findings are discussed.

Hypertension in autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) has been shown to be associated with a greater than 50 percent incidence of hypertension prior to deterioration in renal function as assessed by glomerular filtration rate. The present study provides evidence for increased cardiac pre-load, as assessed by plasma atrial natriuretic factor (ANF) and cardiac index, in hypertensive as compared to normotensive ADPKD. The hypertensive ADPKD patients exhibited an increased renal vascular resistance as compared to the normotensive patients in spite of comparable glomerular filtration rates. It is hypothesized that the renal involvement of hypertensive ADPKD patients causes an impaired renal response to the observed increase in cardiac index, and also may release a venoconstrictor (such as angiotensin) which contributes to the enhanced cardiac pre-load and thus the hypertension.

Plasma osmolality predicts extracellular fluid catechol concentrations in the lateral hypothalamus.

The lateral hypothalamus has an important role in regulating food and water intake. We have investigated the endogenous release of monoamines from the lateral hypothalamus during manipulations of plasma osmolality and circulating volume. Adult male Sprague-Dawley rats implanted with carbon paste in vivo electrochemical (EC) electrodes in the lateral hypothalamus were placed on a 72-h water deprivation schedule. Although the carbon paste EC electrode has an intrinsically ambiguous signal in which changes in ascorbic acid may appear as changes in catechol concentrations, pharmacologic studies in lateral hypothalamus indicated that the electrode most likely measured norepinephrine and possibly epinephrine. On the test day, the EC electrodes were scanned with linear sweep voltammetry from -0.2 to +0.4 V at a rate of 5 mV/s. Semiderivative signal processing showed catechol and hydroxyindole peaks at +0.11 and +0.23 V, respectively. Baseline recordings were made prior to rats drinking distilled water, 10% sucrose, 5% dextrose, 0.30% NaCl, 0.90% NaCl, or 10% d-mannitol. To control for the act of drinking, other implanted dehydrated rats were intraperitoneally injected with 5% dextrose, 0.30% NaCl, or 0.90% NaCl. To dissociate the effects of osmolality and circulating volume on the EC response, hydrated rats implanted with EC electrodes were subcutaneously injected with 12% NaCl or intraperitoneally injected with 35% polyethylene glycol. Other rats subjected to water deprivation and osmotic challenges were decapitated and trunk blood was collected for measurements of plasma osmolality and hematocrit. Similar experiments were conducted using homozygous Brattleboro rats which lack arginine vasopressin (AVP) but which preserve normal plasma osmolality with prodigious drinking.(ABSTRACT TRUNCATED AT 250 WORDS)

Pregnancy: an overfill or underfill state.

With pregnancy, the expansion of plasma and extracellular fluid volume, increased cardiac output, and increased glomerular filtration rate (GFR) and renal blood flow (RBF) are compatible with primary renal sodium and water retention with secondary enlargement of the vascular compartment (overfill hypothesis). Alternatively, a primary enlargement of the vascular compartment (eg, prostaglandin-mediated vasodilation and placental arteriovenous shunting) with secondary renal sodium and water retention (underfill hypothesis) is supported by the activation of the renin-angiotensin-aldosterone (RAA) system, resetting of vasopressin release and thirst to a lower plasma osmolality, and a further stimulation of the RAA system and vasopressin when pregnant rats' water intake is maintained at a level of a virgin rat fluid intake, diminished BP and increased cardiac output in the first trimester, and worsening of hyponatremia with sodium restriction in the pregnant but not the virgin rat. With the underfill hypothesis, normal sodium and water excretion with mineralocorticoid escape could be mediated by hormonally induced increases in GFR and RBF associated with pregnancy. The nature of such a hormone effect of pregnancy to enhance renal hemodynamics remains to be defined.

Diabetes insipidus in pregnancy.

Diabetes insipidus (DI) and pregnancy may coexist and, when they do, present challenging diagnostic and therapeutic problems. Women with preexisting central DI usually experience increased thirst and require additional hormone replacement. Women with nephrogenic DI have an increased water turnover. Of interest is a group of women with transient DI of gestation. In some of these patients, central DI is brought to the fore by increases in water turnover during pregnancy as well as increments in the metabolic clearance of arginine vasopressin (AVP), especially near term. Others have a "vasopressin-resistant" form of the disease, which in one case followed by us appeared to be due to marked increments in circulating cystine-aminopeptidase (vasopressinase). This patient's DI was resistant to pitressin, but she concentrated her urine when given dDAVP. Her vasopressinase levels 2 weeks postpartum were still several-fold those of normal term gravidas. Her DI remitted, and she concentrated her urine appropriately 2 months postpartum. This article reviews the different forms of DI peculiar to pregnancy.