The effect of age on insulin-degrading activity in rat tissue.

Insulin-degrading activity was measured in the 100,000g supernatant fraction of muscle, liver, and kidney from rats of varying ages. Young animals (four weeks old) had the highest activity in all three tissues. By seven weeks of age the activity in both muscle and liver had decreased significantly as compared with four-week-old animals. A slight but nonsignificant decrease occurred in kidney. In animals over one year of age the insulin-degrading activity in all three tissues was significantly less than the activities at either four or seven weeks. In contrast the effect of age on degradation of albumin and parathormone was much less marked.

Insulin binding and degradation by muscles from streptozotocin-diabetic rats.

Insulin degradtion by muscle was examined in normal, streptozotocin-induced diabetic rats, and diabetic rats treated with insulin. Insulin degradation by the 100,000 X g supernatant fractions was identical in all three groups, but insulin metabolism by the intact epitrochlaris muscle was significantly increased in diabetic animals. Insulin treatment of the diabetic animals partially restored the activity toward normal. Specific binding of 125l-insulin to the intact muscles was also increased in the diabetic animals. Streptozotocin diabetes, therefore, increased the binding and degradation of insulin by intact muscle but did not alter the insulin degradation by the total soluble intracellular degradative activity.

Insulin degradation by hepatocytes in primary culture.

The mechanism by which the liver degrades insulin has not yet been completely clarified. In intact, non-"leaky" cells the primary process seems to be mediated by initial receptor binding. We now demonstrate that isolated rat hepatocytes in primary culture are suitable for examining insulin degradation. Hepatocytes did not leak degrading activity into the medium, and thus, the degradation seen was essentially exclusively cell mediated. [125I]Iodoinsulin degradation by these cells was dependent on time and cell concentration. There was a short lag time before degradation products could be detected in the medium. After incubation with the hepatocytes, three peaks of 125I-labeled material could be separated by chromatography on Sephadex G-50. The same three peaks were seen with 125I-labeled material extracted from the cells. When [3H]insulin, labeled exclusively at the B-1 phenylalanine residue, was incubated with the cells, additional peaks of labeled material were recovered from the column. These additional peaks were intermediate in size between insulin and iodotyrosine, suggesting the production of products smaller than insulin but larger than individual amino acids. In order to begin to characterize the subcellular mechanisms for insulin metabolism, the effect of various potential inhibitors on insulin degradation were examined. The most effective inhibitors were N-ethylmaleimide, bacitracin, and Kunitz pancreatic trypsin inhibitor. Chloroquine decreased degradation only 10%, and NH4Cl had no detectable effect. The effect of the inhibitors on the purified insulin-degrading enzyme, insulin protease, was also examined. The purified enzyme responded essentially identically as the intact cells to the various inhibitors. From all these data it would seem that lysosomal degradation of insulin in the hepatocyte may be a relatively minor pathway and the neutral protease may play a major role.

A cell-based drug delivery system for lung targeting: I. Preparation and pharmacokinetics.

A drug-loaded tumor cell (DLTC) system has been developed for lung metastasis-targeting drug delivery. Doxorubicin was loaded into B16-F10 murine melanoma cells (96 microg/10(6) cells). The loading process led to the death of all the carrier cells. The diameter of DLTC was 15.03+/-2.36 microm (mean +/- SD). The amount and rate of doxorubicin being released from the DLTC mainly depended on the drug loading and carrier cell concentration. Over a 6-month storage in phosphate buffered saline (PBS) at 4 degrees C, the decrease in intracellular drug concentration and the carrier cell number were less than 25% and 5%, respectively. After a bolus injection of 30 microg doxorubicin in either DLTC form or free solution into the mice tail veins, drug deposit in the lung from DLTC was 3.6-fold of that achieved by free drug solution. The latter resulted in higher drug content in liver and spleen. Extensive trypsinization of DLTC reduced its lung targeting effect by 30%, and the density of surface adhesion molecule GM3 on DLTC surface by 25%. In conclusion, this DLTC system demonstrated a lung-targeting activity that may be partially attributed to its specific surface characteristics.

Hemodynamic response to vasopressin during V1-receptor antagonism in baroreflex-deficient subjects.

Six quadriplegic subjects and 6 control subjects received high-dose arginine vasopressin (AVP) infusions at rates of 500, 1,000, 2,000, and 4,000 microU.kg-1.min-1 in consecutive 10-min intervals. Six additional quadriplegic subjects received low-dose AVP infusions at rates of 50, 100, 200, 400, and 800 microU.kg-1.min-1. All subjects were studied once with and once without administration of a selective V1-receptor antagonist. During high-dose AVP infusions without V1-receptor blockade, mean arterial pressure (MAP) increased from 80 +/- 4 to 87 +/- 5 mmHg (P < 0.05) in quadriplegic subjects but was unchanged in control subjects. In the presence of V1-receptor blockade, MAP decreased from 75 +/- 4 to 58 +/- 4 mmHg (P < 0.001), and heart rate (HR) increased from 61 +/- 5 to 80 +/- 5 beats/min (P < 0.001) in quadriplegic subjects. In the studies on control subjects, MAP decreased only from 75 +/- 3 to 72 +/- 5 mmHg (P < 0.05), whereas HR increased from 64 +/- 4 to 87 +/- 4 beats/min (P < 0.001). Plasma renin activity (PRA) increased in both quadriplegic and control subjects. The effects of low-dose AVP infusions on MAP, HR, and PRA in quadriplegic subjects were similar to those observed during high-dose infusions. Thus, in the absence of baroreceptor-mediated sympathetic nervous system responses, a vasodilatory effect of AVP that is capable of producing marked reductions in MAP can be demonstrated in the presence of V1-receptor blockade.

Effects of vasopressin V1-receptor blockade during acute and sustained hypovolemic hypotension.

The response of vasopressin and its role in the maintenance of arterial pressure during and after development of hypotensive central hypovolemia were studied in tilt table studies in quadriplegic subjects. The studies were performed during acute head-up tilting to a maximally tolerated degree of tilt (8 subjects) and during sustained head-up tilt following a 20% reduction in mean arterial pressure (MAP) (11 subjects). Studies in all subjects were performed on two separate days, once with and once without administration of a selective vasopressin V1-receptor antagonist. During acute head-up tilting, plasma vasopressin concentrations (PAVP) did not increase significantly until MAP decreased to approximately 60 mmHg at maximal tilt. There was no difference in the degree of hypotension produced in the presence compared with the absence of V1-receptor blockade. There was also no difference in plasma renin activity (PRA) or in plasma cortisol or aldosterone concentrations at maximal tilt. In contrast, during sustained head-up tilt following a 20% reduction in arterial pressures, systolic and mean arterial pressures were significantly lower and PRA was significantly higher in the presence than in the absence of V1-receptor blockade. PAVP increased and was significantly higher after 30 min of sustained tilt than pretilt PAVP in supine posture. These studies do not provide evidence of a role for vasopressin in the maintenance of arterial pressure during the acute development of hypotensive hypovolemia in human subjects, but they do provide evidence of a modest role for vasopressin in the maintenance of arterial pressure when the effect of hypovolemia is more moderate and sustained.

Characteristics of vasopressin release during controlled reduction in arterial pressure.

Because of the interruption of the descending sympathetic nervous pathways, individuals with cervical spinal cord injury experience orthostatic hypotension when in an upright posture. The changes in hemodynamic parameters that occur during upright posture can be closely monitored and quantitated during progressive head-up tilting on a tilt table. We have utilized this method to assess the response of vasopressin and other vasoactive hormones to gradual, progressive reductions in arterial pressure and to identify possible threshold responses to baroreceptor stimulation in human subjects. Studies were performed in 12 quadriplegic subjects, 3 paraplegic subjects, and 3 normal control subjects. Data from the studies in paraplegic and normal subjects did not differ and were pooled as control data. In quadriplegic subjects, mean arterial pressure (MAP) decreased from 93 +/- 4 mm Hg to 60 +/- 3 mm Hg in a closely correlated (r = 0.948, p < 0.002) linear relationship with increasing degrees of tilt, whereas in control subjects, MAP increased from 81 +/- 4 to 88 +/- 3 mm Hg. Plasma vasopressin concentrations (Pavp) increased minimally in quadriplegic subjects until MAP was reduced to levels that were 25% to 30% lower than MAP with subjects in the supine posture. Beyond this level of hypotension, Pavp increased markedly. Log-linear regression analysis of these data showed a highly significant correlation (r = 0.85, p < 0.0002) between in Pavp and MAP, which defines Pavp as an exponential function of decreasing MAP. Changes in Pavp in control subjects were minimal during incremental head-up tilting. In contrast, plasma renin activity (PRA) increased in both quadriplegic and control subjects. Log-linear regression analysis of these data showed highly significant correlates between in PRA and degree of tilt in both quadriplegic (r = 0.958, p < 0.0002) and control (r = 0.873, p < 0.0002) subjects. Plasma atrial natriuretic peptide concentrations decreased linearly with increasing degrees of tilt. The rate of decline in Panp was greater in quadriplegic than in control subjects. These studies provide additional evidence that Pavp increases exponentially as a function of decreasing MAP and suggest that a critical threshold level of hypotension exists at which vasopressin release accelerates rapidly in response to baroreceptor stimulation. At this level of reduced MAP, Pavp reaches levels that are potentially capable of exerting a pressor effect.