GH stimulates sugar transport into diaphragm muscle of hypophysectomized rats, but this effect subsides in 2–3 h and a period of refractoriness follows in which the cells do not respond to further stimulation with the hormone. To determine the briefest period of in vitro GH-tissue interaction required to induce 1) the early stimulatory effect of GH on the transport of 3-O-methylglucose (30MG) and 2) the delayed refractory response, the GH-tissue interaction was interrupted at various times with antibodies against GH. Diaphragms isolated from hypophysectomized rats were incubated with rat GH (rGH), 0.4 μg/ml for 30 min at 0-2 C, to permit binding of the hormone to the cells in the absence of metabolism. Next, the muscles were warmed to 37 C for various times in the presence of rGH, then cooled again to 2 C and exposed for 1 h to either monkey serum or monkey anti-rGH antibodies (ArGH). The muscles were then washed in buffer and again warmed to 37 C for various times. Their ability to transport 30MG was assessed in the final hour at 37 C. To test for refractoriness, additional GH (1 μg/ml) was added during the final h at 37 C. If diaphragms were not warmed to 37 C before the addition of ArGH, no stimulation of 30MG transport or production of refractoriness was obtained. Diaphragms that were warmed to 37 C for 1–10 min and then exposed to monkey serum exhibited both the early stimulatory and the delayed refractory responses to GH. In contrast, diaphragms warmed to 37 C for 1 min with rGH and then exposed to ArGH showed no stimulation of 30MG transport. However, full stimulation of transport was observed if tissues were warmed to 37 C for 10 min before exposure to ArGH; an intermediate response was obtained after 5 min of incubation with rGH at 37 C. These findings indicate that ArGH can neutralize the action of rGH which has associated with tissue receptors, but as metabolism proceeds at 37 C, events are generated which stimulate 30MG transport after the rGH is neutralized. These initial events were not blocked by 5 mM theophylline during the first 10 min at 37 C, although theophylline did prevent the stimulation of transport when added during the final hour, when 30MG transport was assessed. When 10 μg/ml rGH were tested, the initial events resulting in stimulation of transport could be completed by warming tissues for only 1 min before the addition of ArGH. Refractoriness was not produced by the brief exposure to rGH at 37 C that initiated transport. However, if incubation at 37 C proceeded for 180 min with rGH (0.4 μg/ml) before addition of ArGH, refractoriness did develop. Partial refractoriness occurred after tissues were warmed for 90 min after the initial exposure to rGH. A 30-min period of exposure to rGH at 37 C before neutralization of the hormone with ArGH was not sufficient to cause refractoriness. The different periods of metabolism required to initiate the stimulatory effect of GH on sugar transport and to induce refractoriness suggest that these effects of GH are not causally related. © 1979 by The Endocrine Society.
