A nonrecirculatory liver slice perifusion system has been developed and utilized for investigating glucagon-stimulated glycogenolysis in normal and diabetic states. It has been shown here that slices maintained in this system experience a controlled environment with respect to temperature and pH and remain viable throughout a three-hour experimental period based upon their maintenance of intracellular potassium levels. Although glycogen content falls by 40%, slices exhibit significant glycogenolysis in a dose-response manner upon challenge with glucagon, with maximal concentrations eliciting a 2.2-fold stimulation. This system, which permits nonrecirculatory challenge of liver tissue and subsequent analysis of both intracellular events and overall physiological responses, is extremely useful for examining hormonal mechanisms operating for glucagon, particularly at low concentrations. Using this methodology, liver slices challenged with glucagon exhibit a biphasic dose-response for glycogenolysis. While the second phase parallels cAMP (cyclic adenosine 3':5'-monophosphate) accumulation and cAMP-PK (cAMP-dependent protein kinase) activation, the first is mediated independent of cAMP. Trinitrophenylhistidine-1, homoarginine-12-glucagon (THG), which can antagonize glucagon-stimulated adenylate cyclase, exhibits 50% partial agonist activity for cAMP production and cAMP-PK but full agonism for glycogenolysis. Separation between these events is only two-fold indicating a cAMP-mediated process. [Des-amino-fYRKKE]-glucagon, ([Des-amino-His¹,D-Phe⁴,Tyr⁵,Arg¹²,Lys¹⁷·¹⁸,Glu²¹]-glucagon), another adenylate cyclase antagonist, does not stimulate cAMP or cAMP-PK up to 25 μM yet still elicits glycogenolysis. These results demonstrate that glucagon does indeed stimulate both cAMP-independent as well as cAMP-dependent glycogenolysis in normal liver. In diabetic systems, glucagon elicits attenuated adenylate cyclase activity in liver plasma membranes with reduction in basal activity and extent of stimulation. Maximal stimulation of cAMP production is also reduced by half in liver slices, but in both systems (normal vs. diabetic) EC₅₀ values for cAMP production are identical. Neither THG nor [des-amino-fYRKKE]-glucagon stimulate cAMP production or cAMP-PK in diabetic liver slices. While THG lowers blood glucose levels in vivo, [des-amino-fYRKKE]-glucagon acts as an agonist. These results suggest that the mechanisms which operate for glucagon-stimulated glycogenolysis in normal liver are attenuated in the diabetic state. Furthermore, antagonism of cAMP production alone is insufficient to antagonize glucagon's overall physiological action.

Several feeding-related factors can affect taste sensitivity or preferences and therefore may be part of a homeostatic regulatory mechanism. Cholecystokinin (CCK), a hormone which reduces food intake in several species, has also been postulated to interact with the orosensory characteristics of food. To test this hypothesis, the effects of CCK-8 and food deprivation on the short-term intakes of water, sucrose solutions (0.05 to 1.0 M), and saline solutions (0.05 and 0.15 M) were determined. In most cases, CCK (2 μg/kg) reduced sucrose intake when measured either as the amount consumed or the number of licks in a short period (nine minutes). Additionally, CCK reduced the intake of 0.15 M NaCl in satiated rats and water intake in both hungry and satiated rats. Rats usually consumed more sucrose when hungry than when satiated or fed ad libitum; CCK-induced suppression of intake, however, was generally greater in the satiated or ad libitum conditions than in the hungry condition. There was no systematic effect of sucrose concentration on the amount of CCK-induced suppression of intake, which suggests that CCK regulates rather than interferes with ingestion. To determine whether the CCK-induced suppression is due to a change in the peripheral taste signal, the integrated chorda tympani responses to sucrose and NaCl tastes were recorded in rats anesthetized with either urethane, Innovar-Vet, or a combination of urethane and alpha-chloralose. The only significant effect of CCK was the slight increase in the initial response to 0.3 M sucrose after the infusion of a total of 10 μg of CCK-8 into rats anesthetized with Innovar-Vet. In general, therefore, the effect of CCK on sucrose intake does not appear to be due to a peripheral taste change; an analysis of single taste fibers, however, would be more conclusive. An examination of the effects of CCK on central gustatory and reward areas might yet provide a mechanism for the CCK effect on taste-motivated ingestion.

Seven field experiments and two in-vitro studies were performed to elucidate hormonal control of staminate flowering in gynoecious and monoecious buffalo gourd (Cucurbita foetidissima HBK.) sex types. Objectives included development of techniques effecting staminate induction on gynoecious phenotypes which normally produce abortive stamenless male buds. Natural and synthetic growth regulants shown to modify sex expression in other cucurbits were surveyed for their masculinizing potential. Several compounds exogenously-applied to apical meristems elicited changes in shoot morphology. However, only aminoethoxyvinylglycine (AVG, an ethylene synthesis inhibitor) effected staminate induction on gynoecious segregates. Growth rate, patterns of female flowering or ontogeny of stamenless buds differentiated prior to treatment were not influenced by AVG. AVG was applied at various dosages (0-500 ppm) and produced male buds on all replicates treated at levels of 125 ppm or higher. The mean number of staminate buds induced varied linearly with dosage and averaged from 0-7.5 male flowers/shoot. A control model for staminate induction mediated by endogenous ethylene was advanced and potential benefits of this phenomenon to breeding efforts and/or to hybrid seed production were discussed. Ethephon (an ethylene releasing compound) was applied at various dosages to monoecious plants in anticipation of simulating the gynoecious phenotype. Although morphological changes were evident (i.e. reduction in shoot growth rate and floral initiation, increase in floral bud abortion and tissue senescence), ethephon failed to reduce staminate flowering or increase differentiation of antherless buds as expected. Dosage levels employed and/or confounding environmental factors may have contributed to the lack of staminate inhibition. During in-vitro studies, indirect evidence for ethylene-mediated control of male flowering was obtained by staminate proliferation in buds of gynoecious explants treated with silver nitrate (an inhibitor of ethylene action) and by formation of stamenless buds on monoecious explants treated with ethephon. However, low levels of floral induction under culture conditions employed rendered these results inconclusive. An incidental study of segregation ratios among AVG-facilitated self- and cross-pollination progeny upheld the supposition for monogenic inheritance of gynoecy in buffalo gourd.

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