Gh: a GTP-binding protein with transglutaminase activity and receptor signaling function.

The alpha 1-adrenergic receptors activate a phospholipase C enzyme by coupling to members of the large molecular size (approximately 74 to 80 kilodaltons) G alpha h family of guanosine triphosphate (GTP)-binding proteins. Rat liver G alpha h is now shown to be a tissue transglutaminase type II (TGase II). The transglutaminase activity of rat liver TGase II expressed in COS-1 cells was inhibited by the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) or by alpha 1-adrenergic receptor activation. Rat liver TGase II also mediated alpha 1-adrenergic receptor stimulation of phospholipase C activity. Thus, G alpha h represents a new class of GTP-binding proteins that participate in receptor signaling and may be a component of a complex regulatory network in which receptor-stimulated GTP binding switches the function of G alpha h from transglutamination to receptor signaling.

Fluorescent glucagon derivatives. I. Synthesis and characterisation of fluorescent glucagon derivatives.

The synthesis of monofluorescein, monorhodamine, and mono-4-nitrobenz-2-oxa-1,3-diazole (NBD) derivatives of glucagon is reported. The fluorescent groups were introduced by converting tryptophan-25 to 2-thioltryptophan using thiol-specific fluorescent reagents. All derivatives retained the ability to activate adenylate cyclase when compared to glucagon and thus were considered full agonists. IC50 values of 6.8.10(-9), 1.7.10(-8), 1.8.10(-8) and 5.4.10(-9) M were measured in rat liver membranes for NBD-, fluorescein-, rhodamine-Trp25-glucagon and native glucagon, respectively. From the IC50 values Kd values of 2.16.10(-9), 4.10(-9), 2.10(-9) and 1.72.10(-9) M were calculated for the binding of NBD-, fluorescein-, rhodamine-Trp25-glucagon and native glucagon, respectively. The highest quantum yield (0.18) of the monomer derivatives was obtained with fluorescein-Trp25-glucagon in phosphate-buffered saline (pH 7.4). Difluorescein-glucagon was also prepared by reacting the amino groups of histidine-1 and lysine-12 with fluorescein isothiocyanate and dimer derivatives were prepared using fluorescein-labelled 2-thiolTrp25-glucagon. Difluorescein-glucagon bound only weakly to glucagon receptors and displayed antagonist properties. The dimer derivative formed from two difluorescein-2-thiolTrp25-glucagon molecules had similar poor binding qualities, whereas the dimer formed from difluorescein-2-thiolTrp25-glucagon and 2-thiolTrp25-glucagon exhibited, at low concentrations, properties similar to monofluorescein-glucagon. Both dimer derivatives were only sparingly soluble in aqueous medium. Specific binding of fluorescein-Trp25-glucagon and difluorescein-glucagon to rat hepatocytes was followed using flow cytometry.

Transglutaminase II: a new class of GTP-binding protein with new biological functions.

Tissue type transglutaminase (TGase II) is historically a member of the transglutaminase family, which covalently cross-links cellular proteins and polyamines. A recent new finding in the TGase II field is that the enzyme functions as a signal mediator from receptors to an effector in transmembrane signaling. This review will discuss the recent development of TGase II. This new signal transducer was termed Gh when initially discovered and was recently found to be TGase II. To help the reader understand the role of Gh as a signal mediator, the role of heterotrimeric G-proteins in hormone-mediated transmembrane signaling is briefly discussed. We have highlighted how Gh transmits the alpha 1-adrenoceptor signal to the phospholipase C-delta 1 and how Gh is activated and deactivated compared to the prototype of heterotrimeric G-proteins. Recent developments regarding the structure-function of Gh and other biological functions of Gh are discussed to facilitate understanding the impact of Gh in cells.

Quantitative histochemistry of the primate skin. III. Glyceraldehyde-3-phosphate dehydrogenase.

Glyceraldehyde-3-phosphate dehydrogenase activities were assayed with a fluorometric micromethod in various parts of the skin and its appendages. Enzyme activity was abundant, particularly in the epidermis and mucous membrane (4.2 to 11.3 moles/hr/kg dry wt.). The keratin layer of sole epidermis also contained a considerable amount of enzyme activity (2.6 moles/hr/kg dry wt.). A relatively low activity was found in the apocrine and sebaceous glands (1.8 to 3.6 moles/hr/kg dry wt.). The enzyme was influenced by various activators and inhibitors. The activators were 1-cysteine (200% increase), mercaptoethanol (+300%), EDTA (+300%), and the inhibitors, pCMB, Cu++ and Co++ (nearly 100% inhibition). Either arsenate or orthophosphate was an absolute requirement for the enzyme reaction. It is speculated that this dehydrogenase in skin and appendages may be a regulator of the Pasteur effect, hence of glycolysis, as it may be in muscle or in ascites tumor cells.

Evidence for an early free radical-mediated reperfusion injury in frostbite.

Frostbite is characterized by acute tissue injury induced by freezing and thawing. Initial complete ischemia is followed by reperfusion and later, tissue necrosis. These vascular events support the hypothesis that free radical-mediated reperfusion injury at thawing might contribute to tissue necrosis after frostbite in a manner similar to that seen after normothermic ischemia. To test this hypothesis, rabbit ears were frozen at -21 degrees C for 30, 60, 90, or 120 s and rewarmed at room temperature (22 degrees C). Rabbits were treated "blindly" with saline alone, highly purified, pharmaceutical grade superoxide dismutase (SOD), allopurinol, or deferoxamine. The area of ear necrosis was determined 3 weeks after frostbite by "blinded" morphometry. The administration of SOD at the time of thawing significantly improved viability in ears frozen for 60 and 90 s, but not in those frozen for 30 or 120 s. Deferoxamine also improved viability in ears frozen for 60 s. Allopurinol did not significantly affect ear survival. Electron micrographs showed the appearance of severe endothelial cell injury beginning during freezing and extending through early reperfusion. Later, neutrophil adhesion, erythrocyte aggregation, and microvascular stasis were seen. These findings suggest that free radical-mediated reperfusion injury has a role in frostbite, and quantitate the proportion of the injury that is due to this mechanism.

Interactions of pure beta gamma-subunits of G-proteins with purified beta 1-adrenoceptor.

The role of the beta gamma-subunits in the interaction of G-proteins was examined with beta 1-adrenoceptors purified from turkey erythrocytes and pure beta gamma-subunits prepared from turkey erythrocytes and bovine brain. On a non-denaturing polyacrylamide gel, the mobility of beta gamma-subunits was increased when incubated with beta 1-adrenoceptor and the beta 1-adrenergic agonist 1-(-)-isoproterenol, whereas on incubation with the antagonist 1-alprenolol the mobility was unchanged. Furthermore, the beta 1-adrenoceptor was retarded on a Sephadex G-50 column equilibrated with beta gamma-subunits and agonist. No retardation occurred in the presence of antagonist. These data suggest a direct interaction of activated beta 1-adrenoceptors with isolated beta gamma-subunits of G-proteins.

The hormonal regulation of adenylate cyclase.

The regulation of adenylate cyclase by hormones and by GTP regulatory proteins was investigated in native membrane systems and in systems reconstituted from purified components. These studies can be summarized as follows. The stimulatory beta 1-adrenoceptor catalyses the activation of a complex between the GTP stimulatory protein GS and the catalytic unit C. The agonist-receptor complex can activate a few cyclase units in native membrane systems as well as in reconstituted systems. GS from turkey erythrocytes is functionally different from rabbit liver GS, the latter being more amenable to activation by guanyl nucleotides in the absence of hormone. The coupling between the beta 1-adrenoceptor GS and C is efficient when compared with the coupling obtained in native membrane systems. GTP/GDP exchange at the alpha S subunit requires the presence of the beta gamma subunits. A mechanism for the inhibition of adenylate cyclase by the inhibitory GTP regulatory protein Gi is suggested.

The role of oxygen free radicals in mediating the reperfusion injury of cold-preserved ischemic kidneys.

We evaluated the hypothesis that postischemic renal failure is caused primarily at reperfusion by oxygen-derived free radicals in a swine model designed to realistically mimick human cadaveric renal transplantation. Both kidneys were removed, flushed with Euro-Collins solution, stored 24 hr at 4 degrees C, and then transplanted to a second pig. Experiments were paired, each pig receiving one treated and one control kidney. All pigs received the optimal conventional regimen of hydration, phenoxybenzamine, furosemide, and mannitol to allow assessment of free radical treatment superimposed thereupon. Two days later creatinine clearance (CCR) was measured from each kidney via separate ureterostomies. Untreated kidneys developed severe functional impairment, CCR falling from a normal level of 25.5 +/- 6.3 ml/min (n = 8) to 7.7 +/- 0.9 ml/min (n = 14, P less than .05 vs. control). The infusion of 20 mg of the free radical scavenger superoxide dismutase (SOD) into the renal artery at reperfusion substantially ameliorated this injury (CCR = 15.9 +/- 1.7 ml/min, n = 18, P less than 0.05 vs. control). A dose-response curve to SOD showed no effect of doses of 0.2 mg (CCR = 8.0 +/- 1.1 ml/min, n = 4) or 2 mg (CCR = 7.7 +/- 0.9, n = 5), and no greater benefit from 100 mg (CCR = 16.1 +/- 2.1 ml/min, n = 3, P less than 0.05 vs. control). Blocking the generation of superoxide radicals from xanthine oxidase with allopurinol (50 mg/kg) afforded similar protection (CCR = 18.2 +/- 1.8; n = 11, P less than 0.01 vs. control). On the other hand, following an 18-hr period of cold ischemia, little damage was sustained by the untreated (control) kidneys (CCR = 22.1 +/- 0.6 ml/min). Consequently, under these conditions the ablation of free radical generation with allopurinol provided no significant benefit. These findings suggest that after a critical period of cold ischemic preservation, metabolic changes take place within the kidney that lead to free radical generation and consequent tissue injury upon reperfusion, despite optimal preservation by conventional methods. This damage can be prevented by simple nontoxic measures--which, therefore, show great promise for use in the prevention of early renal failure following cadaveric renal transplantation.

Improved survival in free skin flap transfers in rats.

We have demonstrated previously that oxygen-derived free radicals are important mediators of tissue injury in experimental island skin flaps that have been subjected to prolonged ischemia (vascular occlusion) followed by reperfusion. In this study the role of oxygen free radicals in ischemia/reperfusion injury has been investigated in free flap transfers. Groin skin flaps were harvested, stored at room temperature for 21 to 24 hours, and transplanted to the contralateral groin. These free flap transfers normally exhibit a high incidence of complete necrosis. Treatment before the onset of reperfusion with a single dose of superoxide dismutase (SOD), a scavenger of superoxide radicals, increased the survival rate of these skin flaps from 38% in the control group to 76% (p less than 0.025). Tissue levels of SOD were measured before ischemia, after ischemia but before reperfusion, and 30 minutes after reperfusion: untreated flap tissues, which were destined to undergo necrosis, exhibited a significant decrease in SOD activity after reperfusion, whereas SOD-treated flap tissues, destined to survive, demonstrated increased enzyme activity. High levels of tissue SOD activity thus appeared to be associated with improved flap survival. The results have significant clinical implications with regard to organ preservation and transplantation.

The effects of hyperbaric oxygen on free flaps in rats.

The effects of hyperbaric oxygen on survival were investigated in free flaps and island flaps. Skin flaps transplanted following 18, 21, and 24 hours of preservation at 24 degrees C demonstrated survival rates of 20%, 10%, and 0%, respectively. Treatment with hyperbaric 100% oxygen improved the survival rates to 66%, 67%, and 40%. A preservation time of 21 to 24 hours at room temperature appears to be the threshold of irreversible ischemic damage. In acute island flaps, flap survival was improved significantly from 35% to 53% and 64% of the random flap area by preoperative or postoperative treatment, respectively. Prolonged preoperative and postoperative treatment improved survival to 66%.

Effect of electrical stimulation on survival of skin flaps in pigs.

The purpose of this study was to investigate the effect of electrical stimulation on ischemia-induced tissue injury in skin flaps. Bipedicle skin flaps measuring 4 X 20 cm were created bilaterally on the flanks of 12 Yorkshire pigs. The ischemic central portions of the flaps were treated with 35 mA of electrical current at a frequency of 128 Hz for 30 minutes twice daily during the initial nine days following skin-flap elevation. The treatment schedule consisted of negative-electrode stimulation during the first three days, positive-electrode stimulation during the second three days, and negative-electrode stimulation during the seventh to ninth days. Five control pigs underwent either no treatment (n = 3) or sham treatment (n = 2). The mean area of the skin flaps exhibiting necrosis was 28.0% in the control animals and 13.2% in the stimulated animals. These areas were significantly different (p less than .001). The results indicate that pulsed electrical stimulation can improve the survival of skin flaps.

Porcine dermal collagen as a wound dressing for skin donor sites and deep partial skin thickness burns.

Collagen was extracted by pepsin digestion from porcine skin, and collagen membrane was prepared by salt precipitation. The porcine collagen membrane was evaluated as a burn wound dressing in deep partial skin thickness burn wounds in rats. Burn wounds, 4 x 4 cm, were inflicted by exposure of skin to 75 degrees C for 15 s followed by de-epithelialization. Wound healing was assessed by planimetry of epithelialization on day 10 after injury. Open wounds exhibited 24 per cent of wound area re-epithelialized. Collagen membrane dressing significantly improved the healing to 69 per cent of wound area (P < 0.0001). In a completely separate experiment, the porcine collagen membrane was applied as a wound dressing to the donor sites of burn patients, and its effect on wound healing was compared with that of a petroleum jelly gauze dressing. The donor sites covered with petroleum jelly gauze had re-epithelialized by an average of 14.5 days (ranging from 13 to 16 days) after wounding. The wounds dressed with collagen membrane demonstrated a significant increase in the healing rate. Complete re-epithelialization was observed by 10.3 days (ranging from 10 to 12 days) after wounding (P < 0.0001).

Skin flap necrosis in guinea pigs: limitation of glucose supply and accumulation of lactate.

The distribution of glucose and lactate within skin flaps was determined in full-thickness guinea pig skin (excluding the panniculus carnosus). The distal end of the flaps had a low glucose content (less than 50 percent of normal) but high lactate level (2 to 3 times normal) during the 3 days after flap elevation. Progressively decreasing gradients of glucose content, and increasing gradients of lactate content, were demonstrated from the mid-region to the distal end of the flaps at 3 hours postoperatively. The decrease in glucose and the accumulation of lactate may play a significant role in skin necrosis.

Osteoblastic and osteoclastic activation in coronal sutures undergoing fusion ex vivo.

Numerous studies have demonstrated the importance of dura mater in the normal development and regeneration of the cranium and its sutures. The purpose of this study was to analyze the effect of dura mater on the metabolism of bone during the process of premature suture fusion. Previously, coronal sutures of fetal rats have been shown to fuse in serum-free culture after removal of their dura mater, whereas sutures of neonatal rats resist fusion even without their dura mater present. Sutures from these two distinct developmental stages were evaluated by assaying alkaline phosphatase and tartrate-resistant acid phosphatase (TRAP), marker enzymes of bone synthesis and catabolism, respectively. Coronal sutures with adjacent calvaria were dissected from fetal day 19.5 (F19) rats (n = 142) and neonatal day 1 (N1) rats (n = 42) and randomly divided into two groups each: F19 sutures with dura mater intact; F19 sutures with dura mater removed; N1 sutures with dura mater intact; and N1 sutures with dura mater removed. Calvaria were grown in serum-free medium for up to 21 days, and enzyme activities in suture regions were assayed by microanalytical techniques at different time intervals of culture. F19 sutures without dura mater exhibited significant increases in enzyme activities during days 7 to 21 of culture, whereas those without dura mater did not. N1 sutures with or without dura mater exhibited no significant changes in enzyme activities during the 14-day period of culture. The process of F19 suture fusion, occurring in the absence of dura mater, coincided with the increased activities of both alkaline phosphatase and TRAP. These cellular, enzymatic changes may have implications for the cellular events comprising craniosynostosis in vivo.

Metabolic adaptations in delayed skin flaps. Glucose utilization and hexokinase activity.

The distribution of glucose and hexokinase activity was determined in the epithelial tissue of delayed bipedicled skin flaps in guinea pigs. The periods of "delay" were 1, 3, 7, 14, or 21 days. The flap survival was maximal (100% of the flap) when the flap elevation was performed either 7 or 14 days following the "delay" procedure. When the flap elevation was performed 1, 3, or 21 days following the "delay" procedure, the result was partial necrosis. A differential distribution of epithelial glucose was found within the bipedicled flaps. The lowest glucose level (30% of normal) was at a distance of 2 to 3.5 cm from the end of the caudal pedicle during the first day after the "delay" procedure. This decreased glucose content recovered toward normal levels during the later part of the "delay" period. The bipedicled flaps exhibited increased hexokinase activity during the 3-week period of the "delay," and the responses of hexokinase activity and tissue glucose levels to the "delay" procedure were reciprocal in the caudal half of the flaps.

Enzymatic responses to skin flap elevation following a delay procedure.

Alterations in enzyme activities of glucose metabolism were determined in the distal portion of the skin flaps of guinea pigs elevated following 3, 7, 14, or 21 days of the delay period. Hexokinase, pyruvate kinase, lactate dehydrogenase, and glucose 6-phosphate dehydrogenase activities were increased during the delay period, whereas isocitrate and malate dehydrogenases exhibited little alteration. Increases in glycolytic enzyme activities observed during the delay procedure were diminished in the flaps elevated during initial 3 days of the delay period, but were maintained or increased further in the flaps elevated at 7 to 21 days. Despite high levels of enzyme activities during the early period of the delay, the flaps elevated during this period exhibited partial necrosis with a low glucose level and decreased enzyme activities. It is concluded that tissue glucose level and its utilization are crucial factors for flap survival.

Tissue glucose and lactate following vascular occlusion in island skin flaps.

Neurovascular island skin flaps were elevated in the right groin area of Sprague-Dawley female rats weighing from 200 to 250 gm. Survival rates of the flaps were determined following venous and/or arterial occlusions of the femoral vessels. All the flaps exhibited an 80 percent decrease in tissue glucose content and an increase in tissue lactate content 4 to 7 times normal during venous and/or arterial occlusion. In the groups occluded for 8 hours, venous occlusion resulted in loss of all the flaps; arterial occlusion yielded survival of 70 percent of the flaps; and occlusion of both vessels yielded survival of 30 percent of the flaps. The results indicate that venous occlusion is more detrimental to flap survival than arterial occlusion. Surviving flaps exhibited a glucose content of 3.5 times normal, and dying flaps exhibited a glucose content of 20 percent of normal and a lactate content of 5.1 times normal. The ratio of tissue lactate to glucose may serve as an index for tissue viability: normal flaps, 0.3, surviving flaps, 1.2, and dying flaps, 7.5.

Enzymatic activation associated with programmed fusion of the posterior interfrontal sutures in rats.

Fetal rat coronal sutures in culture undergo fusion in the absence of their dura mater. Coinciding with the period of fusion are marked cellular enzymatic changes. Alkaline phosphatase, a marker of osteoblastic activity, and tartrate-resistant acid phosphatase (TRAP), a marker of osteoclastic activity, both increase significantly within fusing sutures and indicate changes in the control of bone synthesis and breakdown. Other enzymes not specifically related to bone formation or degradation also show activation within these fusing sutures. These enzymes include tartrate-sensitive acid phosphatase (TSAP), a marker of lysosomal activity; hexokinase, a glycolytic enzyme; glucose 6-phosphate dehydrogenase (G6PD), an enzyme of the pentose monophosphate shunt; and glutathione reductase, an enzyme of the antioxidant pathway. In the present study, we compared the enzymatic changes previously seen ex vivo with those occurring in vivo during the programmed closure of the posterior interfrontal suture of the rat. This suture fuses between postnatal days 10 and 30 in the rat. The sagittal suture, which remains patent during this period, was used to establish baseline enzymatic activities in a comparable midline suture. Neonatal rats were killed at postnatal days 2, 4, 5, 8, 10, 12, 15, 20, and 30, and posterior interfrontal and sagittal sutures with bone plates on either side were removed. The suture regions of the samples were isolated, dura mater was removed, and suture regions were assayed by microanalytical techniques. Activities of alkaline phosphatase, TRAP, TSAP, hexokinase, G6PD, and glutathione reductase were measured. DNA content was also assayed, and enzyme activities were expressed per amount of DNA. Three pups were killed at each time point, and three to five assays were performed per suture (posterior interfrontal or sagittal) for each time point assayed. Alkaline phosphatase and TRAP activities showed marked increases in fusing sutures compared with nonfusing controls, similar to the increases demonstrated ex vivo. TSAP and hexokinase also showed elevations in the fusing posterior interfrontal sutures, with the greatest differences predominantly during the period of fusion, comparable to the changes seen ex vivo. However, G6PD and glutathione reductase, enzymes of the antioxidant pathway, did not demonstrate the same degree of activation seen ex vivo in fusing sutures. In fact, the levels were actually higher in the patent sagittal samples for the majority of time points examined. Alkaline phosphatase and TRAP activity elevations indicated both osteoblastic and osteoclastic activation during fusion, as seen in the ex vivo phenomenon. TSAP and hexokinase increases also reflected activation in lysosomes and in cellular metabolism during fusion, paralleling the ex vivo situation. However, a less clear pattern of activation in the antioxidant pathway, in contrast to the pattern seen ex vivo, was present. These differences may reflect the different environments of sutures in vivo and ex vivo. Alternatively, oxidative stress may play a more central role in the pathologic process of induced suture fusion ex vivo than in programmed suture fusion in vivo.

Effects of sympathetic denervation and oxygen free radicals on neovascularization in skin flaps.

An island skin flap, with its sole blood supply based on the inferior epigastric vessels, in Sprague-Dawley rats (female, 220 to 250 gm) was used as a model for the investigation of neovascularization. Flap survival after pedicle ligation was considered an indicator of neovascularization. Vascular pedicles were ligated on days 2 to 5 after flap elevation, and the time course of neovascularization in the innervated and denervated flaps was determined by measurements of survival on day 7 after pedicle ligation (on days 9 to 12 postoperatively). Neovascularization sufficient to maintain viability was established at 4 and 5 days after flap elevation in the innervated and denervated flaps, respectively. The effects of various scavengers of oxygen free radicals on neovascularization were evaluated in the innervated and denervated flaps. The pedicles were ligated 3 days after flap elevation. Flap survival was assessed on day 7 after pedicle ligation (on day 10 postoperatively). Treatment with a single dose of deferoxamine (50 mg/kg) increased the viability from 48 to 69 percent of flap area in the denervated flaps (p < 0.01) but produced little effect on viability in the innervated flaps. In the denervated flaps, treatments with a single dose of superoxide dismutase, intravenously and intraarterially, also substantially increased the survival rates from 29 to 86 percent (marginally significant) and 100 percent (p < 0.05), respectively. Allopurinol improved the survival from 43 to 88 percent; the difference was not statistically significant. The results suggest that denervation resulted in a delay of neovascularization and that severe sympathetic denervation contributes to the production of oxygen free radicals, which may exert their inhibitory effects on neovascularization.

Skin-flap metabolism in rats: oxygen consumption and lactate production.

Overall glucose metabolism was evaluated by measuring the rate of oxygen consumption (QO2) and lactate production in the pedicle skin flaps of rats. Skin flaps exhibited increases in QO2 and lactate production in vitro. The distal portion of the flap is characterized by a greater deposition of glucose to lactate during the initial 3 days following flap elevation. The contribution of glycolysis and of the oxidative pathways to glucose metabolism in skin flaps approximates that in normal skin on day 7 postoperatively.