Evidence for delayed development of the glucagon receptor of adenylate cyclase in the fetal and neonatal rat heart.

The effects, in vivo, of epinephrine, glucagon, and dibutyryl cyclic adenosine 3',5'-monophosphate (cyclic AMP) on the glycogen content of rat heart and liver and, in vitro, upon adenylate cyclase activity in homogenates of rat heart and liver were determined during the latter third of gestation and the neonatal period. Hepatic glycogen was depleted by epinephrine, glucagon, and dibutyryl cyclic adenosine 3',5'-monophosphate, but myocardial glycogen was depleted only by epinephrine and dibutyryl cyclic AMP in the neonates. Hepatic adenylate cyclase activity was augmented by both epinephrine (10(-5) M) and glucagon (10(-5) M), and myocardial cyclase was increased only by epinephrine in tissue obtained from 16, 18, and 20 day fetal rats. Myocardial adenylate cyclase responsiveness to glucagon was present in tissue obtained from rats 4 wk of age and older. It is concluded that in contrast to hepatic adenylate cyclase, myocardial adenylate cyclase in the rat is not responsive to glucagon during gestation and that responsiveness to glucagon and the associated ability of glucagon to deplete myocardial glycogen do not develop until well after birth.

Responsiveness to glucagon in fetal hearts. Species variability and apparent disparities between changes in beating, adenylate cyclase activation, and cyclic AMP concentration.

Previous studies of the ability of the immature heart to respond to glucagon have yielded conflicting results. To test the possibility that the apparent discrepancies might be explained in part by species variability, isolated hearts of fetal mice and rats (13-22 days' gestational age) were studied under identical conditions in vitro. Changes in atrial rate and ventricular contractility were measured in spontaneously beating hearts exposed to glucagon, and activation of adenylate cyclase was assayed in cardiac homogenates. In mice of 16 days' gestational age or less, there was no change in heart rate in response to glucagon; at 17-18 days, minimal responsiveness was present; and after 19 days, 10muM glucagon caused an increase in spontaneous atrial rate of 30 +/- 4% (SEM) (P less than 0.001). Measurement of the extent and speed of volume displacement of the isotonically contracting hearts with a specially constructed capacitance transducer revealed that ventricular inotropic responsiveness also appeared after 17-19 days. Cardiac stores of glycogen were reduced in older hearts exposed to glucagon, but not in those aged less than 16 days. In contrast, glucagon failed to activate adenylate cyclase in homogenates of hearts of fetal mice at any age. Furthermore, glucagon failed to elicit an increase in the concentration of cyclic AMP in spontaneously beating hearts that developed tachycardia. Responses in hearts of fetal rats were distinctly different from those in mouse hearts: at no age was there any change in heart rate, strength of contraction, glycogen content, or adenylate cyclase activation. Thus, there are major species differences in cardiac pharmacological maturation. Although the mouse heart develops the ability to increase its rate and strength of contraction and to undergo glycogenolysis in response to glucagon well before birth, the rat heart does not. In addition, there is an apparent disparity in late fetal mouse hearts between the ability of glucagon to induce functional responses and its ability to stimulate adenylate cyclase and increase cyclic AMP levels. It is impossible, of course, to rule out absolutely the possibility that localized increases in a critical cyclic AMP pool were present but too small to measure in the entire tissue. Nevertheless, the most obvious interpretation of our results is that they are compatible with the hypothesis that glucagon may exert some of its hemodynamic effects independently from the adenylate cyclase-cyclic AMP system in the late-fetal mouse heart.

Effects of dibutyryl cyclic adenosine 3',5'-monophosphate and parathyroid extract on calcium and phosphorus metabolism in hypoparathyroidism and pseudohypoparathyroidism.

It has been proposed previously that the metabolic defect in pseudohypoparathyroidism which accounts for parathyroid hormone unresponsiveness is an absence or abnormal form of the adenyl cyclase system in kidney and presumably in bone. To determine whether there is an associated defect in the response mechanism to cyclic adenosine 3',5'-monophosphate (cyclic AMP), the effects of parathyroid extract (PTE), and dibutyryl cyclic AMP were compared in patients with either surgical hypoparathyroidism or pseudohypoparathyroidism. PTE and dibutyryl cyclic AMP both increased serum and urinary calcium, lowered the serum phosphorus, and increased urinary phosphorus in patients with hypoparathyroidism. PTE also increased urinary cyclic AMP in these patients. PTE increased serum and urinary calcium and urinary phosphorus but did not alter serum phosphorus or urinary cyclic AMP in the patients with pseudohypoparathyroidism. Dibutyryl cyclic AMP increased the serum and urinary calcium, lowered the serum phosphorus, and increased urinary phosphorus in all the patients with pseudohypoparathyroidism. The results indicate that (a) dibutyryl cyclic AMP can reproduce the effects of parathyroid hormone on calcium and phosphorus metabolism in man, (b) the response mechanism to cyclic AMP appears to be intact in pseudohypoparathyroidism, and (c) PTE apparently produces some of its characteristic effects on calcium and phosphorus metabolism in pseudohypoparathyroidism in the absence of an increase in urinary cyclic AMP.

Appearances of responses to glucagon in cultured neoatal rat heart cells.

Heart cells from neonatal rats have been cultured. The ability of 10(-5)M glucagon to stimulate adenylyl cyclase activity, and to increase the cAMP concentration and the beating rate in these cells was followed as a function of time in culture. The cultured cells show no response to 10(-5)M glucagon until 5 weeks. By contrast, the cells do respond to 10(-5)M epinephrine with an increase in beat rate, adenylyl cyclase activity and cAMP levels when freshly prepared or after 1 week in culture. Previous studies on the newborn rat heart, acutely isolated, have also shown that the neonatal rat heart is insensitive to glucagon until 4-5 weeks after birth. We conclude that the cultured neonatal rat heart cells can also mature in the same time frame with respect to a glucagon response.

Rate-limiting steps in isoproterenol and forskolin stimulated lipolysis.

Using the flask-incubated fat cell system, effects of isoproterenol and forskolin on glycerol release, cyclic AMP levels and protein kinase were studied. Isoproterenol increased cyclic AMP levels, protein kinase activity and glycerol release over the same concentration range (10(-9) M to 10(-6) M). Forskolin also increased all three variables in a concentration-dependent manner (10(-7) M to 10(-4) M). The maximum response for each variable was significantly greater with forskolin than with isoproterenol. A combination of isoproterenol and forskolin resulted in an additional increase in cyclic AMP over forskolin alone, but no significant increase in protein kinase activity or glycerol release. These results support the concepts that the maximum lipolytic response to isoproterenol is limited by the accumulation of cyclic AMP and the maximum lipolytic response to forskolin is limited by some step distal to cyclic AMP production, possibly activation of protein kinase. At high concentrations of forskolin or with a combination of forskolin and isoproterenol, cyclic AMP levels were in excess of those needed to maximally activate protein kinase and lipolysis.

Role of calcium ion in hormone-stimulated lipolysis.

Using the flask-incubated fat cell system, the effects of Ca2+ removal from the incubation medium on the lipolytic system were studied. The removal of Ca2+ resulted in a total abolition of the lipolytic response and the increased cyclic AMP accumulation produced by ACTH. The lipolytic response to isoproterenol and forskolin were reduced approximately 40% by Ca2+ removal, but cyclic AMP accumulation was not altered in the presence of either of these agents using a Ca2+-free medium. The lipolytic response to the dibutyryl analog of cyclic AMP was also reduced by omission of Ca2+ from the incubation medium. It is concluded the Ca2+ is required for the interaction of ACTH with its receptor and the resultant activation of adenylate cyclase. Ca2+ also is required at some step in the lipolytic process distal to cyclic AMP.

On the lipolytic action of parathyroid hormone in man.

An investigation was carried out to determine whether bovine PTH stimulates lipolysis in human fat tissue, whether this action is mediated by cyclic adenosine 3', 5'-monophosphate and whether the N-terminal 1-34 peptide of bovine PTH is responsible for the lipolytic effect. Studies were also performed to determine if parathyroid extract (PTE) produces lipolysis in normal subjects and in patients with pseudohypoparathyroidism in whom there is a defect in the adenylate system in response to PTH in the renal cortex and presumably in the skeletal system as well. It was found that highly purified bovine PTH in the concentration range between 10(-9) M and 10(-5) M stimulated lipolysis in vitro by human fat in a dose-dependent manner. Significant increases in glycerol production were observed at concentrations of PTH as low as 10(-9) M and maximal increases were seen at 10(-6) M. The hormone significantly increased the concentration of cyclic adenosine 3' ,5'-monophosphate in fat tissue. The synthetic N-terminal 1-34 peptide of bovine PTH was as effective as the native hormone in stimulating glycerol production at a concentration of 10(-9) M-10(-6) M. PTE, 100 mU per kg per min for 30 min given intravenously, produced transient increases in the concentration of plasma free fatty acid in each of eight normal subjects, three patients with hypoparathyroidism and eight patients with pseudohypoparathyroidism. Purified bovine PTH also increased plasma free fatty acid in each of two normal subjects. It is concluded that PTH stimulates lipolysis in human subcutaneous fat, that this action of the hormone is mediated through cyclic adenosine 3', 5'-monophosphate and that the N-terminal 1-34 peptide portion of the hormone is responsible for this lipolytic action. Further, PTE stimulates lipolysis in vivo in man. There appears to be no defect in the adenylate cyclase system in the fat cell in response to PTH in patients with pseudohypoparathyroidism.

The aging Leydig cell: 1. Testosterone and adenosine 3',5'-monophosphate responses to gonadotropin stimulation in rats.

Plasma testosterone levels before and after a single injection of hCG were significantly lower in 24-month old rats than 60--90 day old animals (p less than 0.001). Even with repeated hCG administration for three weeks, plasma testosterone levels of old rats could not be restored to levels present in unstimulated young rats. In response to in vitro LH and 8-bromo-cyclic AMP stimulation, purified young Leydig cells produced significantly higher amounts of testosterone than Leydig cells from old rats. Maximal testosterone formation of the young Leydig cells in response to LH was 42.0 +/- 6.88 ng/10(6) cells, while cells from old rats produced only 16.8 +/- 3.69 ng/10(6) cells (p less than 0.01). However, the dose of LH at which one half maximal response (ED50) occurred was 0.1 mIU/ml for young Leydig cells and 0.05 mIU/ml for old Leydig cells. Basal and 1.0 mIU LH-stimulated cyclic AMP formation were comparable in both groups, but cyclic AMP formation in response to 10 mIU of LH was significantly less in the old rats (p less than 0.05). Present results demonstrate impaired steroidogenic capacity of old rats both in vivo and in vitro. Decreased testosterone response in old rats most likely is the consequence of understimulation of Leydig cells by gonadotropin; however, there appear to be additional intrinsic defects in old Leydig cells.

AANA Journal course: update for nurse anesthetists--low molecular weight heparin: pharmacology and regional anesthetic implications.

Low molecular weight heparins were first introduced in the United States in May 1993 as an alternative to currently available anticoagulant therapy. Like standard heparin, these anticoagulants inhibit activation of a number of coagulation enzymes, but low molecular weight heparins have their primary inhibitory effect on factor Xa. A decrease in plasma protein binding by low molecular weight heparin results in greater bioavailability and a more predictable therapeutic response than that of standard heparin. Although drug action is not measurable by commonly available laboratory tests of coagulation, greater predictability of drug response led to acceptance of these agents for perioperative thromboprophylaxis. The introduction of low molecular weight heparin into the perioperative surgical management of patients also has influenced perioperative anesthetic care. Postmarketing reports of the formation of spinal epidural hematoma when these agents were used concurrently with regional anesthesia prompted the US Food and Drug Administration to issue an advisory to anesthesia providers. This Journal course includes the pharmacology of the class of drugs known as low molecular weight heparins, the incidence and risk factors for the development of spinal or epidural hematoma, and current recommendations for the use of these anticoagulants in conjunction with spinal or epidural anesthesia. Guidelines for the postoperative use of indwelling spinal or epidural catheters in patients who receive this drug therapy in the course of their perioperative care are presented.