Miyuki Honma

An ultrasensitive method for the simultaneous determination of cyclic AMP and cyclic GMP in small-volume samples from blood and tissue

Abstract Taking advantage of the reports of Cailla et al. that succinylation of cyclic AMP and cyclic GMP causes a tremendous increase in their affinities for the antisera (8,9), a simple and ultrasensitive radioimmunoassay has been developed for the simultaneous determination of the cyclic nucleotides in a small volume of biological materials. The binding of succinylated cyclic nucleotides by their specific antibodies is so stabilized in an imidazole buffer that undiluted plasma or the concentrated extract of tissues is directly assayed for cyclic nucleotides without carrying out any prior purification, such as deproteinization of column chromatography. Thus, blood levels of cyclic AMP and cyclic GMP are readily determined in as a small volume of plasma as 50 μl. Periodical changes in the blood concentration of cyclic nucleotides in response to adrenergic and hormonal stimuli were followed in individual rats by means of this method.

Increases in plasma cyclic AMP dependent on endogenous catecholamines

Administration of tyramine (with or without phentolamine) as well as induction of ether anesthesia or insulin hypoglycemia caused a sharp increase in plasma cyclic AMP in rats. Based on the findings that the treatment of rats with reserpine, 6-hydroxydopamine, cocaine or propranolol totally abolished tyramine-induced increases in plasma cyclic AMP, it was concluded that catecholamines released from sympathetic neuronal terminals by tyramine could activate adenylate cyclase via the stimulation of postsynaptic beta-adrenoceptors. In contrast, catecholamines secreted from adrenal medulla were largely responsible for the increase in plasma cyclic AMP induced by ether anesthesia; whereas glucagon, in addition to adrenal catecholamines, played a significant role in hypoglycemia-induced increases in plasma cyclic AMP. Assay of plasma cyclic AMP following these stimuli is very promising as a test for adrenergic activities in experimental and clinical studies.

Plasma cyclic GMP: response to cholinergic agents.

S.c. injections of cholinergic agents, carbachol, methacholine and bethanechol, into fasted rats caused rapid increases in the plasma concentration of cyclic GMP, with a sharp peak at 5--10 min after the injection. Acetylcholine gave rise to a rapid accumulation of cyclic GMP in plasma only when administered together with physostigmine which produced only a slight, if any, potentiation of the action of the cholinesterase-resistant choline esters. Cyclic AMP also increased after these drugs, but only subsequently to the rise of cyclic GMP; the primary action of the cholinergic drugs appeared to be the increase in cyclic GMP. Atropine was effective not only in abolishing the increase in plasma cyclic GMP induced by cholinergic drugs but also in lowering the baseline level of cyclic GMP. It was concluded that the plasma concentration of cyclic GMP could serve as a good parameter of cholinergic activity in rats.

Circadian variations in plasma 3':5'-cyclic adenosine monophosphate and 3':5'-cyclic guanosine monophosphate of normal adults.

Abstract Circadian variations in plasma cyclic AMP and cyclic GMP were studied in thirteen male subjects (20–22 years old) under controlled invironmental condition. Plasma collections were made every six hours. Cyclic AMP and cyclic GMP were determined by radioimmunoassay. Individual values of plasma cyclic AMP at 0800 are between 13.0 and 25.8 pmole/ml, and cyclic GMP between 2.5 and 7.0 pmole/ml. Cyclic AMP demonstrated the circadian variation with the maximum level at 1400 and the minimum at 0200, and cyclic GMP with the highest level at 1400 and the lowest level at 0800.

Plasma cyclic nucleotide responses to methacholine and epinephrine in patients with migraine.

SYNOPSIS

Plasma cyclic nucleotide responses to insulin-induced hypoglycaemia and methacholine in patients with hyperthyroidism

Abstract. The effect of insulin‐induced hypoglycaemia and methacholine on plasma cAMP and cGMP levels was studied in normal volunteers, hyperthyroid and hypothyroid patients. A significant positive correlation existed between the maximal increase in plasma cAMP and the maximal decrease in plasma glucose in normals during insulin‐induced hypoglycaemia. Therefore, the plasma cAMP response is considered to be dependent on the degree of hypoglycaemia, rather than the dose of insulin. The cAMP response to hypoglycaemia was significantly higher in hyperthyroid patient, and was lower in patients with hypothyroidism than in normals. The cAMP response of the hyperthyroid patients was normalized when their hyperthyroidism was controlled after 3 months of treatment. The plasma level of cGMP was slightly elevated during hypoglycaemia, but there was no significant difference between controls and hyperthyroid patients. The cGMP response to methacholine, which is probably mediated by cholinergic receptors, was significantly potentiated in hyperthyroid patients. The cAMP response, which is presumably dependent on endogenous catecholamines secreted during metha‐choline‐induced hypotension, was also enhanced in hyperthyroid patients. It is likely that β‐adrenergic receptor responses and cholinergic receptor responses are both enhanced in hyperthyroidism.

Anomalous plasma cyclic AMP responses to glucagon in patients with liver disease.

The purpose of the present study is to show anomalies of the plasma cAMP response of patients with hepatic disorders to a single injection of a low dose of glucagon (1 μg/kg body wt). The response was markedly blunted in patients with liver cirrhosis and potentiated in patients with acute or chronic hepatitis. This glucagon test is, therefore, promising for development as a simple diagnostic means without undertaking liver biopsy to distinguish cirrhosis from chronic hepatitis.

Plasma Cyclic Nucleotide Responses to Psychological Stress in Patients with Schizophrenia

The plasma level of cyclic nucleotides [cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP)] h, humans increases in response to exercise (Lin 1978; Okada et al 1980), to several hormones (Ball et al 1972; Hamet et al 1975; Brodows et al 1976) and to psychalogical stress such as the mirror-drawing test (MDT) (Okada et al 1983). In neurotic patients the responses of plasma cAMP and cGMP have been cbserved to differ fi~m controls (Okada et al 1983). The purpose of the present study is to examine whether plasma cyclic nucleotide responses to MDT are different in schizophrenics than in normal subjects and possibly in pahents with other psychiatric illnesses.

Plasma cyclic 3′,5′-guanosine monophosphate and cyclic 3′,5′-adenosine monophosphate response to methacholine in man

Cholinergic agents are known to induce increases in tissue and plasma levels of cyclic GMP in experimental animals. We observed that i.m. injection of methacholine, a cholinergic agent, caused significant increases in plasma cyclic GMP and cyclic AMP in man.

Plasma cyclic nucleotide responses to methacholine in patients with Adie's syndrome

Abstract The purpose of the present study was to show anomalies of the plasma cyclic nucleotide responses of patients with Adies syndrome to an intramuscular injection of methacholine (80 μg/kg body wt). The methacholine-induced increase in plasma cGMP was much larger in patients with Adies syndrome than in controls. There was also a small but significant rise of plasma cAMP in patients with Adies syndrome, while no change was observed in controls, following the methacholine challenge. It is likely that cholinergic receptor responses are enhanced in patients with Adies syndrome, in good agreement with the postulated pupillary postganglionic denervation in affected pupils.