New methods for the isolation of skeletal muscle sarcolemma and sarcoplasmic reticulum allowing a comparison between the mammalian and amphibian beta(2)-adrenergic receptors and calcium pumps.

New methods were established for the rapid and simultaneous isolation of multiple sarcolemmal and sarcoplasmic reticular fractions from very small amounts (0.25-2.0 g) of skeletal muscle. Thebeta(2)-adrenergic receptor and calcium transport systems were used as indices of purity and functional integrity as well as being the focal points of the study. These methods were found to be suitable for the special needs of small tissue samples, allowed rapid preparation and were appropriate for skeletal muscle from various species, frogs to mammals. The sarcolemmalbeta(2)-adrenergic receptor was expressed in frogs and mammals at similar levels of expression (336-454 fmol. x mg(-1)). The calcium pump was also present in sarcolemmal and sarcoplasmic reticular fractions in all species but notable species differences were found. In sarcolemmal fractions, while calcium binding was uniformly low (<1 nmol. x mg(-1)), oxalate stimulation was variable: low in frogs ( approximately 1.05-fold) high in mammals (120-450-fold). In sarcoplasmic reticular fractions, calcium binding was low in frogs (4-9 nmol. x mg(-1)) and much higher in mammals (322-383 nmol. x mg(-1)); oxalate stimulated calcium transport to a much greater extent in frogs (<70-fold) than in mammals (1.6-2-fold). It is concluded that thebeta(2)-adrenergic receptor appears to be strongly conserved in skeletal muscle while the use of calcium pumps evolves from reliance in Amphibia on the sarcoplasmic reticular calcium pump to the use in Mammalia of calcium pumps from both the sarcoplasmic reticulum and the plasma membrane.

Characterization of sarcolemma and sarcoplasmic reticulum isolated from skeletal muscle of the freeze tolerant wood frog, Rana sylvatica: the beta(2)-adrenergic receptor and calcium transport systems in control, frozen and thawed states.

In freeze tolerant wood frog Rana sylvatica, the freeze-induced liberation of glucose plays a critical role in survival in response to sub-zero temperature exposure. We have shown that the glycaemic response is linked to selective changes in the expression of hepatic adrenergic receptors through which catecholamines act to produce their hepatic glycogenolytic effects. The purpose of the present study was to determine if skeletal muscle, another catecholamine-sensitive tissue with glycogenolytic potential, displayed similar or different changes. In order to achieve these objectives, skeletal muscle derived from Rana sylvatica was studied in control, frozen and thawed states. In isolated sarcolemmal fractions, freezing effected an 88% decrease in beta(2)-adrenergic receptor expression but was without effect on the calcium pump; while thawing resulted in a recovery of the beta(2)-adrenergic receptor to 60% of control levels and a 2.4-fold increase in calcium transport. In isolated sarcoplasmic reticular fractions, freezing effected a 52% decrease in calcium binding and a 92% decrease in oxalate-stimulated calcium uptake; while thawing elicited partial normalization to control levels to 70% with respect to calcium binding and to 47% with respect to calcium uptake. Freezing and thawing were associated with increases and decreases, receptively, in blood glucose levels but were without effect on skeletal muscle glycogen content. Thus these muscle changes in Rana sylvatica in freezing and thawing are not linked to glycogen breakdown, are different from those previously seen in liver, and may provide a role in recovery of muscle function during thawing by protecting glycogen stores for contraction and maximizing extracellular calcium for excitation-contraction coupling in the frozen state. The involvement of thyroid hormone in triggering these muscle changes is discussed.

Hepatic changes in the freeze-tolerant turtle Chrysemys picta marginata in response to freezing and thawing.

Select hepatic changes in the freeze-tolerant hatchling turtle, Chrysemys picta marginata, were studied in response to freezing at -2.5 degrees C and thawing. Upon freezing, a small, selective increase in the liver weight with no increase in body weight was seen suggestive of an hepatic capacitance response. In all turtles studies, lobular differences in the hepatic content of glycogen were evident: the smaller lobe contained twice as much glycogen as the larger lobe. The response to freezing and thawing was comparable. Total hepatic glycogen levels of turtles were reduced approximately 60 per cent from control levels in the frozen state and recovered to >80 per cent of control levels in the thawed state. Compared to the control state, turtle blood glucose levels were: unchanged after 12 h in the cool state; reduced 28 per cent after 24 h and increased two-fold after 48 h in the frozen state; and increased 4.5-fold in the thawed state. Thus, changes in hepatic glycogen metabolism occur without large changes in blood glucose levels. In turtle liver plasma membranes, the hepatic alpha(1)-adrenergic receptor was barely detectable and did not change. The beta(2)-adrenergic receptor was expressed at high levels and, compared to control levels, was: unchanged after 12 h in the cool state; reduced 20 per cent after 24 h and 40 per cent after 48 h in the frozen state. On thawing, this receptor was 50 per cent of control levels. While catecholamines working through the beta(2)-adrenergic receptor may effect early hepatic glycogen breakdown in response to freezing, other factors must be involved to complete the process. The plasma membrane-bound enzyme gamma-glutamyltranspeptidase displayed a different pattern of changes indicative of selective modulation: it was increased 2.7-fold over control levels in the cool state; unchanged in the frozen state; and increased 1.8-fold in the thawed state. The activity of the kidney enzyme was decreased in the cool state and slightly increased in the frozen and thawed states emphasizing the tissue-specific nature of the changes in the activity of gamma-glutamyltranspeptidase in response to freezing and thawing. The similarities and differences of the hepatic changes in response to freezing and thawing in the freeze-tolerant hatchling turtle to those we have previously reported for the freeze-tolerant frog are discussed.

Neonatal STZ model of type II diabetes mellitus in the Fischer 344 rat: characteristics and assessment of the status of the hepatic adrenergic receptors.

The Fischer 344 rat was found to be extremely sensitive to the diabetogenic effects of neonatally injected streptozotocin (STZ): injection of 40-100 mg/kg STZ at 1.5 days postnatal produced in the adult graded levels of hyperglycemia in males but not the females. The optimal dose in the 1.5 day old male was 80 mg/kg: it produced hyperglycemia without affecting growth or thyroid status in the adult. The neonatally STZ-injected adult rat displayed characteristics consistent with type II diabetes: mild hyperglycemia accentuated by fasting or consumption of a high fat diet; little change in insulin levels; slight elevation in glucagon levels; no alterations in ketones. Using radioligand binding techniques to isolated rat liver plasma membranes, compared to the control state, the type II diabetic state was found to have: no effect on either alpha(2)- or beta-adrenergic receptor binding; a decrease in the major dominant alpha(1)-adrenergic receptor, reflecting a decrease in receptor numbers but not their affinity; an increase in the plasma membrane calcium transport system, potentially depleting intracellular calcium stores essential for producing an alpha(1)-adrenergic receptor response. Since the alpha(1)-adrenergic receptor-calcium effector system is critical for the actions of catecholamines in the rat, these results suggest that the liver in the type II diabetic state may be refractory to the actions of catecholamines. We propose that the diabetes-evoked decrease in the dominant adrenergic receptor-effector system through which catecholamines act may be the cellular expression of defective glucocounterregulation in the diabetic state.

Evaluation of an optimal temperature for brain storage in delayed 2, 3,5-triphenyltetrazolium chloride staining.

This paper presents the determination of an optimal temperature for delayed 2,3,5-triphenyltetrazolium chloride (TTC) staining. Twenty-one rats were subjected to right middle cerebral artery embolic stroke and sacrificed 96 h following ischemia. The brains were harvested and stained immediately after sacrifice or stored for 8 h at 21-23 degrees C or 4 degrees C, respectively. The stained sections were scanned and infarct volume calculated. The quality of staining, distinction of borders between infarcted and non-ischemic tissue and ease of differentiating ischemic tissue in colored and grayscale images was assessed. The present study indicates that results of TTC obtained immediately after animal sacrifice, or delayed TTC staining while storing the brains at room temperature or 4 degrees C are comparable.

Brain gamma-glutamyltranspeptidase: characteristics, development and thyroid hormone dependency of the enzyme in isolated microvessels and neuronal/glial cell plasma membranes.

The characteristics, cellular locus and regulation of the enzyme gamma-glutamyltranspeptidase (gammaGT) in brain were examined. In rat brain homogenates, the activity of the enzyme exhibited tissue differences--kidney>>>brain==testis>>liver>>skeletal muscle=ventricular muscle and regional differences--brain stem>hippocampus=cerebellum>cerebral cortex, with no significant species/strain differences in the select group of mammals studied. Methods were developed for the isolation from brain of microvessels (MV) and plasma membranes from neuronal/glial cells (N/G PM) utilizing morphological indicators and marker analyses. GammaGT activity was >12 higher in MV than N/G PM; however the enzyme displayed: stability, heat-activation and inhibition with maleate to the same extent in both fractions. A comparative study indicated that in the N/G PM fraction, gammaGT activity was low in all animals studied; gammaGT activity in MV however, was barely detectable in amphibians and reptiles, very low in birds and very high in mammal -mirroring the phylogenetic development of a functional blood-brain barrier. In the rat, gammaGT in both MV and N/G PM displayed a pronounced postnatal increase in activity but the extent and the patterns were different--in all cases, that of the MV greatly exceeded that of the N/G PM and in the MV, the enzyme activity the exhibited the same pattern as the postnatal development of the blood-brain barrier. The induction of congenital hypothyroidism by propylthiouracil (PTU) had no effect on gammaGT in N/G PM but effected a one third reduction in the activity of gammaGT in MV. The normalization by thyroid hormone replacement indicated that MVgammaGT is under thyroid hormone control. The induction of hypothyroidism by PTU in the adult, however, was without effect on enzyme activity in either fraction. The implications of the thyroid hormone dependency of MVgammaGT in the neonatal period and the relationship of gammaGT to the function of the blood brain-barrier is discussed.

Hypothyroidism-evoked shifts in hippocampal adrenergic receptors: implications to ischemia-induced hippocampal damage.

Hypothyroidism was induced in a group of male Fischer 344 rats by administration of 0.05% propylthiouracil (PTU) in the drinking water for 12 weeks. Control rats were not treated. Plasma levels of thyroid hormones indicated that PTU treatment had produced severe thyroid hormone deficiency. In PTU-treated rats compared to control rats, levels of total T3 and total T4 were reduced 54.5% and 53.7%; while levels of free T3 and free T4 were reduced 87.1% and 96.5%. Functional hypothyroidism was demonstrated by: (i) a 49.1% decrease in hepatic plasma membrane alpha1-adrenergic receptor binding, and (ii) a 11.2-fold increase in hepatic gamma-glutamyltranspeptidase activity; relative to the expression of these parameters in control rats. Membranes were isolated from hippocampi of control, PTU-induced hypothyroid and thyroxine-replaced rats and specific adrenergic receptor binding determined by radioligand binding techniques. Hypothyroidism resulted in a shift in the balance of alpha1 and beta2 adrenergic receptor binding by evoking: an increase in alpha1-adrenergic receptor binding to 1.57-fold of control levels; and, a decrease in beta2-adrenergic receptor binding to 64% of control levels. Thyroid hormone replacement carried out in PTU-treated hypothyroid rats at 30 microg/kg s.c. per day for the last 3 days of the 12 week PTU-treatment protocol, which reversed physical and functional hypothyroidism, reversed the observed changes in hippocampal adrenergic receptor binding, indicating them to be thyroid hormone, and not PTU, -dependent. This receptor shift evoked by hypothyroidism may, in part, explain the protective effect of hypothyroidism on ischemia-induced hippocampal damage by favoring inhibitory input and limiting excitotoxic input by catecholamines.

Characterization of gamma-glutamyltranspeptidase in the liver of the frog: 3. Response to freezing and thawing in the freeze-tolerant wood frog Rana sylvatica.

The freeze tolerant wood frog Rana sylvatica was studied to determine the impact of the freezing and thawing of this frog on the activity of gamma-glutamyltranspeptidase in the liver. On exposure to -2.5 degrees C, for 1, 12 and 24 h, frogs were found to be cool, covered with ice crystals and frozen, respectively. Thawing for 24 h at 4 degrees C recovered the frogs completely. A 45 per cent decrease in the liver weight: body weight ratio was notable after 1 h at -2.5 degrees C, suggestive of an early hepatic capacitance response. A glycemic response to freezing was observed: blood glucose levels exhibited a 55 per cent decrease after 1 h at -2.5 degrees C on cooling; a 10.5-fold increase after 12 h at -2.5 degrees C on the initiation of freezing; and a 22-fold increase after 24 h at -2.5 degrees C in the fully frozen state. Blood glucose levels remained elevated four-fold in the thawed state. Plasma insulin levels were increased twofold in the frozen state and 1.8-fold in the thawed state, while plasma ketone levels were increased 1.8-fold in the frozen state and 1.5-fold in the thawed state. Plasma total T3 levels were decreased by 22 per cent in the frozen state and normalized on thawing. In homogenates and plasma membranes isolated from the livers of Rana sylvatica, the activity of gamma-glutamyltrans-peptidase was found to be elevated at all stages of the freeze-thaw process. After 1, 12 and 24 h at -2.5 degrees C, activities were increased 2.5-, 2.3-, 2.4-fold respectively in the homogenates and 2.5-, 2.2-, 2.4-fold respectively in the plasma membranes. After thawing, activities were still increased 1.9-fold in both homogenates and plasma membranes. In homogenates prepared from the kidneys of Rana sylvatica, the activity of gamma-glutamyltranspeptidase was increased 1.4-fold after 1 h at -2.5 degrees C after which it returned to normal. The role of thyroid hormone in producing the increase in gamma-glutamyltranspeptidase in the liver of Rana sylvatica in response to freezing is discussed as is the significance of the enzyme increase in terms of hepatic cytoprotection and freeze tolerance.

Alterations in hepatic adrenergic receptor status in Rana sylvatica in response to freezing and thawing: implications to the freeze-induced glycemic response.

In Rana sylvatica, freeze-induced liberation of glucose from hepatic glycogen stores plays a critical role in conferring freeze tolerance. To determine whether an alteration in hepatic adrenergic receptor status, which dictates catecholamine-directed hepatic glycogenolytic responses, is involved in the glycemic response to freezing, hepatic alpha 1, alpha 2, and beta 2 adrenergic receptors and calcium transport were characterized by radioligand and radioisotopic techniques, respectively, in plasma membranes isolated from the livers of control, -2.5 degrees C-exposed, and frozen-thawed frogs. The three adrenergic receptors display marked and different patterns of changes in response to freezing, with two distinct receptor shifts clearly evident. In the control state, the beta 2 adrenergic receptor dominates over the alpha 1 receptor. At 12 h, beta 2 adrenergic receptor dominance intensifies by a receptor shift involving a decrease in the alpha 1 and alpha 2 adrenergic receptors. Coincident with the initiation of the glycemic response, this early shift may be causally related to it. At 24 h, the alpha 1 adrenergic receptor dominates, achieved by a receptor shift involving a decrease in the beta 2 adrenergic receptor and an increase in the alpha 1 and alpha 2 adrenergic receptors. This shift may be related to the maintenance of the glycemic response. Receptor shifts are associated with changes in calcium transport, which accentuate them. The thawed state is characterized by recovery of alpha, but not beta 2, receptor expression correlatable with, and perhaps allowing, a switch to hepatic glycogenesis. The role of thyroid hormone, whose levels are lower in the frozen state, in inducing receptor shifts is discussed.

Characterization of gamma-glutamyltranspeptidase in the liver of the frog: 2. Response to season, temperature and thyroid hormone in Rana pipiens.

The impact of season and temperature on frog liver gamma-glutamyltranspeptidase was assessed by measuring the activity of this enzyme in plasma membranes isolated from the livers of Rana pipiens obtained as summer and winter frogs; subjected to short-term (3 weeks) temperature acclimation; and subjected to multiple-temperature shifts. Plasma levels of T3 were determined. gamma-Glutamyltranspeptidase was found to be 2.2-fold higher in the summer frog relative to the winter frog; decreased by 44 percent in the summer frog by cold acclimation and increased by 1.7-fold in the winter frog by warm acclimation; and increased by 1.9-fold in the summer frog and 2.8-fold in the winter frog subjected to multiple-temperature shifts. Plasma T3 levels were found to be 42-fold higher in the summer frog relative to the winter frog; decreased by 42 percent by cold acclimation and increased by 2.9-fold by warm acclimation; and decreased by 39 percent and 38 percent in the summer and winter frogs subjected to multiple temperature shifts. T3 replacement during the last phase of the multiple-temperature shift protocol, restored the plasma T3 levels to 75 percent of the control levels and prevented the increase evoked by the multiple-temperature shifts in gamma-glutamyltranspeptidase activity. Indeed, enzyme activity in the T3 replaced state was 19 percent lower than in the control state. The involvement of thyroid hormone as a negative regulator of enzyme activity is discussed.

The impact of type I diabetes on rat liver gamma-glutamyltranspeptidase.

The impact of type 1 diabetes mellitus on liver gamma-glutamyltranspeptidase, a premalignant marker, was studied. Diabetes was induced in male Sprague Dawley and Fischer 344 rats by administration of Streptozotocin, which produced a stable and moderately severe diabetic state. In liver homogenates, gamma-glutamyltranspeptidase was increased over control levels: 1.2, 8.1 and 13.2 fold in Sprague-Dawley rats; 4.8, 58.4 and 84.7 fold in Fischer 344 rats; at 1, 3 and 6 weeks following Streptozotocin treatment. In plasma membranes isolated from the livers of Fischer 344 rats, gamma-glutamyltranspeptidase was increased over control levels: 5.6, 75 and 127 fold at weeks 1, 3 and 6 following Streptozotocin treatment. The relative specific activity of 5'-nucleotidase was found to be similar: 9-14, indicating comparable degrees of plasma membrane purity. Plasma glutamate-pyruvate transaminase levels were minimally and similarly affected at all time points indicating lack of association of increasing gamma-glutamyltranspeptidase activity with overt liver damage. Thyroid hormone replacement, with both T3 (0.6 micrograms/Kg) once a day and T4 (6.0 micrograms/kg) twice a day for three days elicited a further 30% increment in enzyme activity. Insulin replacement (20-40 units/200 g body weight) twice a day for five days reduced enzyme activity 51% at week 6. This was associated with an increase in gamma-glutamyltranspeptidase in the plasma from 14 fold over control levels in the diabetic state at week 6 to 53 fold over control levels after insulin replacement at week 6. It is proposed that the diabetes-induced increase in gamma-glutamyltranspeptidase is reduced by an insulin-directed shedding of the enzyme into the plasma.

Characterization of gamma-glutamyltranspeptidase in the liver of the frog: 1. Comparison to the rat liver enzyme.

The characteristics of the enzyme gamma-glutamyltranspeptidase were determined in frog liver and compared to those of the rat. In Rana pipiens, tissue distribution studies indicated the order of activity to be: kidney >>> liver >> nerve > egg > lung > heart > skeletal muscle in homogenates. In the Rana pipiens relative to the Fischer 344 rat, the activity of the liver enzyme was somewhat greater (1.8-fold) and the kidney enzyme substantially less (25-fold). Frog liver gamma-glutamyltranspeptidase displayed strain-dependent differences in activity with Rana pipiens and Rana sylvatica exhibiting comparable activities and Xenopus laevis exhibiting 20-fold lower activities. No influence of sex was apparent in Rana pipiens in contrast to the sex dependent differences observed in the Fischer 344 rat: female:male = 7:1. In homogenates and plasma membrane fractions of Rana pipiens, Xenopus laevis and the Fischer 344 rat, high, and comparable relative specific activities, were observed, 8-11, coupled with protein yields of 2.2-2.5 per cent indicating the enzyme to be plasma membrane bound and associated with the sinusoidal surface of the liver cell.(ABSTRACT TRUNCATED AT 250 WORDS)

An extended developmental study of gamma-glutamyltranspeptidase in rat liver plasma membranes: identification of specific patterns of changes in activity in the adult as well as the neonatal state.

Homogenates and plasma membranes were isolated from the livers of male Fischer 344 rats ranging in age from 19 hr to 92 days postnatal. These plasma membranes exhibited comparable levels of purity: protein yields were 2-2.5%; relative specific activities of 5'-nucleotidase and ouabain-sensitive Na+/K(+)-ATPase were from 8-11 and from 12-19, respectively. 5'-nucleotidase and ouabain-sensitive Na+ K(+)-ATPase displayed distinct and different developmental patterns. The activity of gamma-glutamyltranspeptidase was found to be at exceptionally high levels in isolated plasma membranes immediately after birth and to decline precipitously thereafter achieving and maintaining low levels from days 3-21 postnatal. Liver plasma membrane gamma-glutamyltranspeptidase activity was observed to increase 9.2 fold from this low point, first rising on day 21, peaking on day 40 and returning to low levels by day 56. From day 56 day to 92 postnatal, gamma-glutamyltranspeptidase activity was expressed at a uniformly low level but a level 2 fold higher than that preceding the rise at day 40. The hormone determinants of these developmental changes in gamma-glutamyltranspeptidase activity are discussed.