Erythropoietin upregulates angiotensin receptors in cultured rat vascular smooth muscle cells.

OBJECTIVE: Plasma renin is not elevated in recombinant human erythropoietin (rhEPO)-induced hypertension but angiotensin converting enzyme inhibitors reduce blood pressure in both human and animal studies. Since rhEPO elevates renin and angiotensinogen messenger RNAs in angiotensin II target tissues such as the aorta, we explored the actions of rhEPO on renin-angiotensin system-related gene transcription of cultured rat vascular smooth muscle cells. DESIGN AND METHODS: To separate direct actions of rhEPO from those mediated secondarily by potential activation of the renin-angiotensin system, vascular smooth muscle cells were cultured with rhEPO and enalapril to inhibit the angiotensin converting enzyme and losartan to inhibit angiotensin II type 1 receptors. RESULTS: Vascular smooth muscle cells cultured with rhEPO (6-8 units/ml) demonstrated elevations (40-120%) in messenger RNAs of the renin-angiotensin system (renin, angiotensinogen, angiotensin receptor types 1 and 2) and increased levels of several messenger RNAs known to respond to angiotensin II (transforming growth factor-beta, insulin-like growth factor-II, epidermal growth factor, c-fos and platelet-derived growth factor). In contrast, cells cultured in the presence of rhEPO and enalapril or losartan showed elevations of messenger RNA for only the two types of angiotensin II receptor. This increase was higher than that obtained when cells were cultured with rhEPO or either antagonist alone. The increase in specific binding of angiotensin II to cells cultured in the presence of rhEPO and enalapril or rhEPO and losartan paralleled the changes in receptor messenger RNA. CONCLUSIONS: rhEPO exerts its primary action on vascular smooth muscle cells via an increase in angiotensin receptor messenger RNA, resulting in a parallel increase in angiotensin II receptor expression. We suggest that increased receptor expression secondarily mediates the expression of other renin-angiotensin system messenger RNAs, which leads to angiotensin II-responsive gene transcription. The elevation in angiotensin II receptors, as observed in response to rhEPO, may provide a mechanism by which other forms of renin-dependent hypertension are initiated.

Paracellular phosphate absorption in rat colon: a mechanism for enema-induced hyperphosphatemia.

The mechanism of colonic phosphate absorption is not well defined. We measured unidirectional phosphate fluxes across rat distal colon epithelium in the absence of transepithelial electrochemical gradients. Steady-state mucosal-to-serosal flux (Jms) was not different from the serosal-to-mucosal flux (Jsm), generating no net flux (Jnet = Jms - Jsm, was not different from "0'). Simultaneous fluxes of mannitol, a paracellular probe, exhibited an identical flux pattern, suggesting that phosphate flux across the colonic epithelium may be mediated through the paracellular pathway. Tight junction permeability was increased with mucosal addition of taurodeoxycholate (TDC, 2 mM) which caused a prompt increase in transepithelial conductance from 7.03 +/- 0.35 to 13.88 +/- 0.35 mS/cm2 (p < 0.001). This was associated with an increase in Jsm, but no change in Jms, for mannitol, resulting in a net flux in the secretary direction. Identical TDC-induced changes were observed in phosphate fluxes, again suggesting phosphate permeation through the intercellular, mannitol pathway. A significant correlation was observed between the permeability of phosphate and the permeability of mannitol, measured both in the mucosal-to-serosal and the serosal-to-mucosal directions and under both control and experimental (mucosal TDC) conditions. Thus, colonic phosphate transport is mediated through the paracellular pathway and enema with high phosphate concentrations (1,760 times blood concentration), can trigger rapid and massive phosphate absorption through this diffusive pathway.

Angiotensin-converting enzyme inhibition in the treatment of renal transplant erythrocytosis. Clinical experience and observation of mechanism.

Recent observations indicate that angiotensin-converting enzyme (ACE) inhibition corrects renal transplant erythrocytosis (RTE). The mechanism for this association is not known. We examined the effect of ACE inhibition on hematocrit, erythropoietin (EPO), and renin substrate. ACE inhibition has been reported to suppress renin substrate, which is known to stimulate EPO and erythropoiesis. In 15 patients with RTE, hematocrit dropped from 52.8 +/- 0.6 (SEM) to 45.8 +/- 1.4% after 8 weeks of treatment with Enalapril, 2.5-20 mg/day. Serum EPO (normal range: 9-30 mU/ml) was high in one, normal in seven, and low in seven patients. ACE inhibition reduced EPO in patients with initial high or normal levels but induced no change in patients with initial low levels. ACE inhibition had no significant effect on renin substrate. In one patient who rejected his first graft, erythrocytosis recurred following a second, successful transplant. Treatment was discontinued because of cough in two patients and symptomatic drop in blood pressure in one patient. We conclude RTE is not caused by hypererythropoietinemia. In patients with normal circulating EPO, erythrocytosis may result from an increase sensitivity to EPO, and ACE inhibition lowered hematocrit by further reduction of this hormone. However, the finding of erythrocytosis in half our patients with suppressed EPO, suggests the participation of non-EPO-mediated mechanism(s). The recurrence of RTE in a patient after a second transplant raises the additional possibility of patient-specific factors in the pathogenesis of this disorder. In contrast to other reports, we documented side-effects (cough, hypotension) in three (20%) of our patients. Our clinical experience, coupled with prior reports of spontaneous resolution of RTE in some patients, suggests that intermittent courses of ACE-inhibition may be the optimal strategy in the use of this form of therapy for RTE.

Analysis of segmental phosphate absorption in intact rats. A compartmental analysis approach.

Available information supports the dominance of the proximal intestine in inorganic phosphate (Pi) absorption. However, there is no strategy for analyzing segmental Pi absorption from a spontaneously propelled meal in an intact animal. We propose a solution using compartmental analysis. After intragastric administration of a 32P-labeled Pi liquid meal containing a nonabsorbable marker, [14C]polyethylene glycol (PEG), rats were killed at 2, 10, 20, 30, 60, 120, and 240 min. The gastrointestinal tract was removed and divided into seven segments, from which 32P and [14C]PEG were recovered. Data was expressed as a percentage of the dose fed, i.e., (32P[in segment] divided by 32P[fed]) and [14C]PEG[in segment] divided by [14C]PEG[fed]), respectively. A compartmental model was constructed and the rate constants for intersegmental transit and segmental absorption were estimated. The "goodness of fit" between the simulated model and the actual data indicates the estimated rate constants reflect in vivo events. The duodenum, with the highest transit and absorption rates, accounted for a third of the total absorption. However, the terminal ileum, with a lower absorption rate but a longer transit time, absorbed an equal amount of Pi. This approach allows the analysis of the mechanism and the regulation of Pi absorption under more authentic in vivo conditions.

Bile salts and ileal calcium transport in rats: a neglected factor in intestinal calcium absorption.

Ileum displays little active transcellular calcium (Ca2+) absorption but is credited with the bulk of Ca2+ absorbed in vivo. We examined the effect of taurodeoxycholic acid (TDC, 2 mM), a bile salt, on mannitol (MN, a marker of intercellular solute traffic) and Ca2+ fluxes in rat ileum. In the absence of electrochemical gradients between the mucosal (M) and serosal (S) bathing media in an Ussing chamber, net flux (Jnet) was observed in the S-to-M direction for both MN and Ca2+, i.e., the unidirectional secretory S-to-M flux (Js-->m) exceeded the absorptive M-to-S flux (Jm-->s). Mucosal TDC caused simultaneous increase in transepithelial conductance and Js-->m for both MN and Ca2+. This was followed by even greater increases in MN and Ca2+ Jm-->s, so that ultimately Jm-->s equaled Js-->m in each case. In control tissue, Js-->m for Ca2+ appeared to permeate exclusively through the intercellular MN pathway while part of Jm-->s for Ca2+ appeared to traverse through a non-MN route. After the TDC-induced increase in intercellular solute permeability, both Ca2+ fluxes appeared to traverse through the aqueous MN conduit. During the postprandial state, the presence of bile salts and the relative abundance of Ca2+ in ileal lumen can cause bulk Ca2+ absorption through the intercellular pathway.

Rapid hypertensinogenic effect of lead: studies in the spontaneously hypertensive rat.

Chronic lead exposure may cause hypertension in normotensive rats. This hypertensinogenic effect has been attributed to perturbations in the renin-angiotensin axis, the contractile response of the vascular smooth muscle, or the intracellular Ca2+ homeostasis as a consequence of the inhibition of Na(+)-K(+)-ATPase activity. In this study we examined the short-term effect of lead exposure on blood pressure, plasma renin activity, vascular contractility, and renal Na(+)-K(+)-ATPase activity and abundance in the spontaneously hypertensive rat. Our data indicate that modest lead exposure caused blood pressure elevation within two weeks in this rat strain that is genetically susceptible to the development of hypertension. This rapid blood pressure-elevating effect did not appear to depend on the mechanisms described in hypertension associated with more chronic lead exposure listed above. This acute model provides an additional approach to the study of lead-induced hypertension.

Influence of recombinant human erythropoietin on blood pressure and tissue renin-angiotensin systems.

In humans, blockade of the renin-angiotensin system with angiotensin converting-enzyme inhibitors (ANG CEI) prevents the rise in blood pressure associated with the administration of recombinant human erythropoietin (rhEPO). This study was conducted to determine whether rhEPO elevates blood pressure in normal Wistar rats and whether the renin-ANG system is affected. Groups of 10 rats each were given rhEPO, ANG CEI (enalapril), rhEPO + ANG CEI, or vehicle. Renin and/or renin substrate mRNA was measured in aortas, kidney, and heart; renin activity (PRA), inactive renin, and renin substrate were measured in plasma. rhEPO raised blood pressure in the normal rat without changing the plasma renin system. ANG CEI prevented this blood pressure rise. Renin-specific mRNA was increased by rhEPO in renal tissue, and renin substrate mRNA was significantly elevated in the kidney and aorta. mRNA for renin and renin substrate were not altered in the heart. In both aorta and kidney, a significant correlation was observed between renin substrate mRNA and blood pressure. The data indicate that rhEPO modulates specific tissue renin-ANG systems, which may contribute to blood pressure elevation.

The importance of non-vitamin D-mediated calcium absorption.

In rapidly growing neonatal rats, the intestine is insensitive to vitamin D, and Ca absorption is solely mediated through a non-energy-dependent process. Changes in Ca absorption associated with pregnancy and lactation are qualitatively similar in vitamin D-replete and vitamin D-deplete rats. Moreover, in vivo studies in man and the rat have demonstrated that the bulk of Ca absorption is accomplished in the ileum, a segment with limited capacity for active Ca absorption and is relatively insensitive to the action of 1,25-dihydroxyvitamin D. In patients with intestinal bypass operations the degree of Ca malabsorption and bone mineral loss is proportional to the length of ileum, not duodenum or proximal intestine, removed. Bile salts and lactose are examples of agents which can augment vitamin D-independent ileal Ca absorption through the intercellular pathway.

Vitamin D-independent regulation of calcium and phosphate absorption.

Convincing evidence for the stimulatory action of 1,25-dihydroxyvitamin D (1,25(OH2)D) on transcellular absorption of calcium (Ca) and inorganic phosphate (P) has led to the consensus that this hormone is the major regulator of Ca and P absorption. Careful review of the literature, however, suggests important regulation of Ca and P absorption by factors and agents other than those mediated by vitamin D. Thus, in rapidly growing neonatal rats, the intestine is insensitive to vitamin D and Ca absorption is entirely mediated through passive mechanisms. Patterns of change in Ca absorption associated with pregnancy and lactation are identical in vitamin D-replete and vitamin D-deplete rats. The presence of active Ca and P absorption in young, growing rats rigidly deprived of vitamin D and of active Ca and P secretion in mature rats optimally replete with vitamin D, also suggests the participation of non-vitamin D factors in the regulation of intestinal Ca and P absorption. The possibility that Ca and P in the peri-enterocyte environment may regulate their own absorption is discussed. Kinetic analysis of 1,25(OH2)D-induced transport mechanisms indicates that saturation would occur at low substrate concentrations, thus raising the question whether these mechanisms would have major regulatory roles under normal dietary conditions. There is also suggestive evidence indicating that even under conditions of low dietary Ca or P intake, the adaptive changes in intestinal absorption may not be mediated by vitamin D alone. Bile salts, lactose and prolactin are discussed as examples of agents which can stimulate Ca and/or P absorption through vitamin D-independent mechanisms.

Forskolin refractoriness. Exposure to the diterpene alters guanine nucleotide-dependent adenylate cyclase and calcium-uptake activity of cells cultured from the rat aorta.

Cells with the morphological properties of endothelial cells were cultured from the rat aorta. The cultured cells accumulated 45Ca2+ from the medium in a manner which was stimulated by forskolin and by 8-bromo-cyclic AMP. Pretreating the cultures for 20 h with forskolin diminished forskolin-dependent Ca2+-uptake activity. Adenylate cyclase activity of cultured cell homogenates was stimulated by guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) and forskolin, and by isoprenaline in the presence, but not in the absence, of guanine nucleotide. p[NH]ppG increased forskolin sensitivity and caused a leftward shift in the forskolin dose-response curve. Pretreating the cultured cells with forskolin for 20 h, conditions that decreased forskolin-dependent Ca2+ uptake, increased basal and guanine nucleotide-dependent adenylate cyclase activity, but not forskolin-dependent activity determined in the absence of p[NH]ppG. Forskolin pretreatment diminished p[NH]ppG's capacity to increase forskolin sensitivity, but did not have a significant effect on either the sensitivity of adenylate cyclase to p[NH]ppG or its responsiveness to isoprenaline. These results suggest that the Ca2+-uptake mechanism is cyclic AMP-dependent and that guanine nucleotides mediated forskolin-dependent cyclic AMP production by the intact cells. In addition, there may be different guanine nucleotide requirements for hormone-receptor coupling and forskolin activation.

Interaction of prostaglandin E2 and beta-adrenergic catecholamines in the regulation of uterine smooth muscle motility and adenylate cyclase in the rat.

Prostaglandin E2 (PGE2) increased the force of the spontaneous contractions of the rat myometrium and decreased the sensitivity of the uterus to the relaxing effects of the specific beta-adrenergic catecholamine agonist isoprenaline. Prostaglandin E2, at concentrations above 10 mumol/l, increased cyclic AMP production by intact muscle strips. The muscle strips were far more sensitive, however, to the inhibitory effect PGE2 had on isoprenaline-dependent cyclic AMP production (threshold less than 0.001 nmol/l). Both PGE2 and isoprenaline stimulated adenylate cyclase activity of a myometrial subcellular (particulate) fraction in a guanyl nucleotide-requiring manner. When present in saturating concentrations (100 mumol/l), the stimulatory effects were not additive, suggesting that the receptors for the two agonists competed for the same catalytic subunit of adenylate cyclase or for the same guanyl nucleotide-requiring factor which couples receptors and enzyme. If muscle strips were incubated with PGE2 before the preparation of the adenylate cyclase-containing particulate fraction, the enzyme became less responsive to stimulation by guanyl nucleotide and by isoprenaline and PGE2 in the presence of guanyl nucleotide. The PGE2 receptor may therefore interact with the beta-adrenoreceptor to inhibit isoprenaline-dependent cyclic AMP production by intact muscle cells by desensitizing adenylate cyclase, possibly at the level of the guanyl nucleotide-dependent coupling step.

Adenylate cyclase activation. Characterization of guanyl nucleotide requirements by direct radioligand-binding methods.

Particulate adenylate cyclase preparations from rat uterine smooth muscle had a single class of [3H]guanyl-5'-yl imidodiphosphate ([3H]GMP.P(NH)P)-binding sites with all of the properties of the guanyl nucleotide-requiring enzyme activation sites (N) which couple hormone receptors and catalytic subunits. These sites bound the radioligand in a reversible manner at low temperature (less than 2 degrees C) but irreversibly at temperatures between 6 and 24 degrees C, properties characteristic of the activation of the enzyme by treatment with GMP.P(NH)P described previously (Krall, J. F., Leshon, S. C., Frolich, M., and Korenman, S. G. (1981) J. Biol. Chem. 256, 5436-5442). Temperature affected the number but not the apparent affinity (Kd approximately 1.0 microM) of binding. The time course of the transition from reversible to irreversible binding was coincident with the irreversible activation of the catalytic subunit. The methyl analog of GTP, guanyl-(beta, gamma-methylene)-diphosphate, a poor irreversible activator compared to GMP.P(NH)P in this enzyme system, was a competitive inhibitor of [3H]GMP.P(NH)P binding but with a 10-fold lower affinity (Kd approximately 10 microM). Using these direct radioligand-binding methods, both an inactive (NL) and active (NL*) form of the guanyl nucleotide-binding activation site were demonstrated, and the transition NL leads to NL* was identified as the temperature-dependent event in catalytic subunit (C) activation. The nondissociability of specific [3H]GMP.P(NH)P binding and the irreversible nature of catalytic subunit activation suggest that a complex of NL*.C characterizes the fully activated state of the smooth muscle enzyme.