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We studied the relationships among serum triacylglycerol (TG), fat pad weight, and lipolytic response to norepinephrine (NE) in iron-deficient rats. We used male Sprague-Dawley International Golden Standard rats. The rats were randomly divided into four groups: two iron-adequate groups for 1 week (1A) and 5 weeks (5A), and two iron-deficient groups for 1 week (1D) and 5 weeks (5D), based on the AIN-93G diet. Iron-deficient treatment caused a significant decrease in hemoglobin (Hb) and hematocrit (Hct) values and an increase in relative heart weight in 1D and 5D rats. Although serum TG was not affected by the 1-week iron-deficient treatment, it was significantly increased by 5-week iron-deficient treatment. The 1-week iron-deficient treatment significantly decreased the relative weight of the retroperitoneal fat pads, but not that of the epididymal fat pads. On the other hand, the 5-week iron-deficient treatment significantly decreased the relative weight of both fat pads; the degree of decrease was 41% and 32% for retroperitoneal and epididymal fat pads, respectively. Basal lipolysis significantly decreased in the epididymal adipocytes from 1D rats, whereas lipolytic response to NE markedly increased. No effect due to the 5-week treatment on basal lipolysis was observed in either retroperitoneal or epididymal adipocytes. In addition, lipolytic response to NE significantly increased in the retroperitoneal, but not the epididymal adipocytes. These results demonstrate that the effects of an iron-deficient diet on fat pad weight are different, depending on the duration of the treatment and the location of fat pads. In addition, iron deficiency-caused hypertriacylglycerolmia may be predominantly related to the increase in lipolysis in retroperitoneal rather than in epididymal adipocytes. The data further show that the increase in lipolysis of epididymal adipocytes occurs in the earlier stage prior to a severe iron-deficient state.

We investigated intracellular Ca2+ regulation in pancreatic acinar cells from rats with diabetes induced by a single injection of streptozotocin (80 mg/kg). Experiments were performed 2 days and 7 days after the injection of streptozotocin. The density of muscarinic receptors, measured by [3H]N-methyl scopolamine binding, was unchanged in 2-day-diabetic rats, but was significantly increased in 7-day-diabetic rats. The percentage of high affinity receptors (RH) and low affinity receptors (RL) determined from the competitive curves with [3H]N-methyl scopolamine and carbachol was not change in 2-day-diabetic rats compared to controls, whereas 7-day-diabetic rats showed a decrease in %RH and an increase in %RL. The carbachol-evoked initial peak of intracellular Ca2+ concentration ([Ca2+]i) was increased in 2-day-diabetic rats and decreased in 7-day-diabetic rats, compared to controls. In the carbachol-induced sustained phase in [Ca2+]i, the response in 7-day-diabetic rats was significantly decreased; however, there was no difference between controls and 2-day-diabetic rats. Carbachol (100 microM)-induced [3H]inositol 1,3,4-trisphosphate generation was significantly lower in diabetic rats than in the controls. The addition of inositol 1,4,5-trisphosphate (1,4,5-IP3) significantly increased 45Ca2+ release from saponin-permeabilized cells in 2-day-diabetic rats, but did not do so in 7-day-diabetic rats. Ca2+ refilling into the intracellular stores, determined by second cholecystokinin-8 (10 nM) stimulation after 10 microM carbachol stimulation, was increased in 2-day-diabetic rats and decreased in 7-day-diabetic rats. These observations indicate that the alterations in intracellular Ca2+ regulation accompanied by changes in transmembrane signaling occur in the earlier stage of the diabetic state. The findings also suggest that the increase in the carbachol-evoked [Ca2+]i peak in 2-day-diabetic rats is related predominantly to the higher sensitivity of 1,4,5-IP3-sensitive Ca2+ stores and the increase in the capacity of Ca2+ refilling in these animals, whereas the reduction in the [Ca2+]i peak in 7-day-diabetic rats appears to be related to the essential decrease in receptor-mediated 1,4,5-IP3 generation and the decrease in Ca2+ refilling capacity.

1. The recovery of rat parotid beta-adrenergic receptors (beta-AR) and adenylate cyclase (AC) from heat (33 degrees C)-induced desensitization was studied. 2. Down-regulated cell surface beta-AR and AC activity in response to isoprenaline (IPR) returned to the control level 120 hr after the termination of heat exposure. 3. However, beta-AR in parotid crude membranes increased over the control level for 48-120 hr. 4. Coupling between beta-AR and G protein(s) was attenuated at 120 hr. 5. These data suggest that beta-AR on the cell surface, but not those internalized, can transduce biological responses.

We studied the relationship between phosphoinositide hydrolysis, phosphatidylcholine hydrolysis, and sn-1,2-diacylglycerol (DAG) formation in response to carbachol stimulation in rat parotid acinar cells. Previously, we demonstrated that DAG formation stimulated with 1 microM carbachol was biphasic: the first peak occurred at 5 min and the second one at 20 min. It was also demonstrated that the second peak was regulated in part by a calmodulin/protein kinase C-dependent mechanism. Based on the kinetic analysis of DAG formation and [32P]phosphoinositide breakdown, the first peak of carbachol (1 microM)-stimulated DAG accumulation was found to be related to the breakdown of [32P]phosphatidylinositol 4-monophosphate ([32P]PIP) and [32P]phosphatidylinositol 4,5-bisphosphate ([32P]PIP2). The second peak was found to be related to [32P]PIP2 breakdown. Carbachol stimulated the release of [3H]phosphocholine into the medium, indicating that the predominant pathway for phosphatidylcholine hydrolysis was via phospholipase C. Moreover, carbachol stimulated the release of [3H]choline metabolites in a time- and dose-dependent manner. This agonist slightly stimulated the release of [3H]ethanolamine metabolites. A calmodulin/protein kinase C-dependent mechanism was also studied and was found to be involved in carbachol-stimulated phosphatidylcholine hydrolysis; W-7, a calmodulin inhibitor and staurosporine, a protein kinase C inhibitor, inhibited the carbachol (1-microM)-induced release of [3H]choline metabolites at 20 min in a dose-dependent manner, but did not have inhibitory effects at 5 min. These results suggest that the first peak of DAG accumulation induced by carbachol is predominantly associated with the breakdown [32P]PIP and of [32P]PIP2 and that the second peak is predominantly associated with [32P]PIP2 breakdown and phosphatidylcholine hydrolysis.

The mechanisms underlying the ability of substance P, to stimulate the sn-1,2-diacylglycerol (DAG) formation were studied using rat parotid acinar cells. During a 60 s stimulation, 1 microM substance P caused a rapid rise in DAG accumulation at 5 s, whereas a low (0.1 microM) concentration of agonist did not. During long term stimulation for 30 min, DAG accumulation induced by 1 microM substance P reached near maximal levels at 5 min and remained elevated for at least 20 min. In contrast, DAG formation induced by 0.1 microM substance P exhibited a peak at 5 min, gradually declined and returned to near basal levels at 30 min. Furthermore, DAG accumulation in response to substance P at 5 and 20 min increased in a dose-dependent manner. The breakdown of both [32P]phosphatidylinositol 4-monophosphate ([32P]PIP) and [32P]phosphatidylinositol 4,5-bisphosphate ([32P]PIP2) stimulated by 1 microM substance P significantly increased from 5 to 20 min and returned to basal levels by 30 min; however, the breakdown of [32P]PIP2 was greater than that of [32P]PIP. At a low concentration of substance P, [32P]PIP2 breakdown reached maximal levels at 5 min followed by a progressive decrease and returned to basal levels at 30 min, whereas the breakdown of [32P]PIP reached maximal levels at 5 min and returned to near basal levels at 10 min. Both concentrations of substance P caused some [32P]phosphatidylinositol breakdown at 5 min. Changes in [3H]inositol trisphosphate induced by substance P were similar to those in [32P]PIP2. In addition, substance P (1 microM) did not stimulate the release of [3H]choline or [3H]ethanolamine metabolites into the medium. Substance P-induced DAG formation was not inhibited by staurosporine, a protein kinase C inhibitor. These results suggest that DAG formation caused by substance P is closely associated with the hydrolysis of phosphatidylinositides but not that of phosphatidylcholine or phosphatidylethanolamine, and is not regulated by protein kinase C-dependent mechanism(s).

The kinetics of carbachol-induced sn-1,2-diacylglycerol (DAG) formation and the underlying mechanism(s) involved in parotid acinar cells were investigated. Supramaximal concentrations of carbachol for amylase secretion (10 microM) caused a transient rise in DAG levels at 10 s. In contrast, this rapid rise was not elicited by 1 microM carbachol, which is the maximally effective concentration for amylase secretion. Carbachol (10 microM) also increased DAG levels linearly up to 20 min, which were sustained for up to a further 10 min. DAG formation stimulated by 1 microM carbachol was biphasic; the first peak was observed after 5 min and the second after 20 min. DAG formation induced by 0.01-0.1 microM carbachol was concentration-dependent and monophasic, peaking at 5 min. The second peak evoked by carbachol was partly inhibited by Ca2+ deprivation from the extracellular space, whereas the first peak was not. Similar results were obtained in experiments using Ca2+ antagonists such as verapamil and LaCl3. The protein kinase C inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) and staurosporine, and a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), significantly inhibited the second DAG peak produced by 1 microM carbachol, but did not alter the first peak. The degree of inhibition of the second peak by these antagonists was comparable. Furthermore, the inhibitory effect of staurosporine and W-7 was concentration-dependent. The A23187-induced accumulation of DAG also was abolished by both staurosporine and W-7. These data indicate that a protein kinase C-dependent mechanism(s) is involved in mediating the second DAG accumulation peak induced by 1 microM carbachol and is mainly regulated by the Ca(2+)-calmodulin complex.