Commentary

Mechanism of inverse regulation of alpha₁- and beta-adrenergic receptors

α₁-Adrenergic receptors (α_1A, α_1B, and α_1D) are regulators of systemic arterial blood pressure and blood flow. Whereas vasoconstrictory action of the α_1A and α_1D subtypes is thought to be mainly responsible for this activity, the role of the α_1B-adrenergic receptor (α_1BAR) in this process is controversial. We have generated transgenic mice that overexpress either wild type or constitutively active α_1BARs. Transgenic expression was under the control of the isogenic promoter, thus assuring appropriate developmental and tissue-specific expression. Cardiovascular phenotypes displayed by transgenic mice included myocardial hypertrophy and hypotension. Indicative of cardiac hypertrophy, transgenic mice displayed an increased heart to body weight ratio, which was confirmed by the echocardiographic finding of an increased thickness of the interventricular septum and posterior wall. Functional deficits included an increased isovolumetric relaxation time, a decreased heart rate, and cardiac output. Transgenic mice were hypotensive and exhibited a decreased pressor response. Vasoconstrictory regulation by α_1BAR was absent as shown by the lack of phenylephrine-induced contractile differences between ex vivo mesenteric artery preparations. Plasma epinephrine, norepinephrine, and cortisol levels were also reduced in transgenic mice, suggesting a loss of sympathetic nerve activity. Reduced catecholamine levels together with basal hypotension, bradycardia, reproductive problems, and weight loss suggest autonomic failure, a phenotype that is consistent with the multiple system atrophy-like neurodegeneration that has been reported previously in these mice. These results also suggest that this receptor subtype is not involved in the classic vasoconstrictory action of α₁ARs that is important in systemic regulation of blood pressure.

The efficient and effective use of hepatocytes from larger species and rare human material requires a reliable storage method for cells not needed on the day of preparation. Cryopreservation would seem to be the only viable alternative. In this study the suitability of a published cryopreservation technique on dog, monkey and human hepatocytes has been examined and the cells were tested for functionality directly after thawing and subsequent to culture using steroid metabolism and hormone responsiveness of glycogen phosphorylase a. Monkey and human hepatocytes appear to survive the freezing and thawing process better than dog cells—the latter losing the ability to respond to adrenergic stimuli and their ability to maintain steroid metabolism in culture. Although monkey and human cells do preserve their steroid metabolising capacity after freeze/thawing, there is not the significant increase in enzyme activity seen during culturing freshly isolated cells. It would appear, therefore, that some damage has occurred to the cells during the freeze/thaw process. As previously noted, Williams’ medium E is superior to Ham’s F-10 in maintaining enzyme activities in culture. It is suggested that cryopreservation is the way forward for the development of stockpiles of viable hepatocytes for biomedical and toxicological research and development but that further modifications to the process are still necessary to optimise the maintenance of liver-specific functions in the thawed cells.

Changes in α₁-adrenoceptor (α₁AR) gene expression in the rat liver during different phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). Septic rats exhibit two metabolically distinct phases: an initial hyperglycemic phase (9 h after CLP, early sepsis) followed by a hypoglycemic phase (18 h after CLP; late sepsis). The [³H]prazosin binding studies show that the density of α₁AR was increased by 30% during the early phase while it was decreased by 24% during the late phase of sepsis. Western blot analyses reveal that α₁AR protein level was elevated by 48% during early sepsis but was decreased by 55% during late sepsis. Northern blot analyses depict that the steady-state level of α_1bAR mRNA was enhanced by 21% during the early phase but was declined by 29% during the late phase of sepsis. Nuclear run-off assays show that the transcription rate of α_1bAR gene transcript was increased by 76% during early sepsis while it was decreased by 29% during late sepsis. The actinomycin D pulse-chase studies indicate that the half-life of α_1bAR mRNA remained unaffected during the early and the late phases of sepsis. These findings demonstrate that during the early phase of sepsis, the increase in the rate of transcription of α_1bAR gene paralleled with the elevations in the α_1bAR mRNA abundance and α₁AR protein level, while during the late phase of sepsis, the decrease in the rate of transcription of α_1bAR gene coincided with the declines in the α_1bAR mRNA abundance and the α₁AR protein level in the rat liver. These observations indicate that the altered expression of α₁AR genes in the rat liver during the progression of sepsis was regulated transcriptionally.

Changes in the distribution of β-adrenoceptors (βARs) in the plasma membrane and the light vesicle fractions of rat liver during different phases of sepsis were studied using [³H]dihydroalprenolol binding and photoaffinity labeling with [¹²⁵I]iodocyanopindolol diazirine. Sepsis was induced by cecal ligation and puncture (CLP). Septic rats exhibit an initial hypermetabolic (hyperglycemic) phase (9 h after CLP; early sepsis) followed by a hypometabolic (hypoglycemic) phase (18 h after CLP; late sepsis). The radioligand studies show that in the plasma membranes, the density of βARs was decreased by 28–32% and 46–69% during the early and the late phases, respectively, of sepsis. In the light vesicles, the density of βARs was increased by 25–30% and 30–35% during the early and the late phases, respectively, of sepsis. The total number of the receptor binding sites (the sum of that in plasma membrane plus light vesicle) was decreased by 11–12% and 21–35% during the early and the late phases, respectively, of sepsis. These results indicate that βARs were progressively internalized from surface membranes to the intracellular sites and, furthermore, they were underexpressed in the rat liver during the progression of sepsis. Since hepatic glucose metabolism is known to be regulated by catecholamines, in part, through βAR mediation, an internalization/underexpression of hepatic βARs may play a role in the altered glucose homeostasis during sepsis, particularly in the late hypometabolic phase of sepsis.

The mechanism whereby somatostatin (SS) produces beneficial effects in established pancreatitis induced by pancreaticobiliary duct ligation (PBDL) is still not clear. The aim of the work was to evaluate the possibility of a direct action of SS on pancreatic acinar cells from rats with acute pancreatitis. For this purpose, we studied the SS-receptor-adenylate cyclase system in pancreatic acinar membranes from both, control rats and rats with experimentally induced acute pancreatitis. On the other hand, it has been reported that cholecystokinin (CCK) diminishes the number of SS receptors in pancreatic acinar cells. Proglumide, a CCK receptor antagonist reduces the severity of acute pancreatitis in the rat. Therefore, we have also examined the effect of proglumide on the somatostatinergic system in controls and rats with acute pancreatitis. Fourteen hours after PBDL, the SS receptors, the capacity of the SS analogue SMS 201–995 to inhibit forskolin-stimulated adenylate cyclase activity and PTX-catalyzed [³²P] ADP-ribosylation of the α₁ subunits of Gi proteins could not be detected in pancreatic acinar membranes. One month after reopening the closed pancreaticobiliary duct (PBD), the pancreas showed regeneration of acinar cells, and the above-mentioned parameters were significantly lower than in the control group. Two months after reopening the closed PBD, all these parameters had returned to control values. The administration of proglumide (20 $\text{mg}\text{kg}$ i.p.), a cholecystokinin receptor antagonist, accelerated pancreatic regeneration and approached all these parameters to control values one month after reopening the closed PBD. The present study suggests that the beneficial effects of SS on established pancreatitis induced by PBDL may not be due to a direct action of the peptide on pancreatic acinar cells at least at 14 hours after PBDL. In addition, these findings suggest that in established pancreatitis the effect of proglumide on the SS receptor-adenylate cyclase system could be due to its action on pancreatic regeneration.

Fibromyalgia, a chronic condition of widespread pain, stiffness, and fatigue, has proven unresponsive to drugs, the use of which is based on the ‘serotonin-deficiency hypothesis’. An alternative hypothesis—failed transcription regulation by thyroid hormone — can explain the serotonin deficiency and other objective findings and symptoms of euthyroid fibromyalgia. Virtually every feature of fibromyalgia corresponds to signs or symptoms associated with failed transcription regulation by thyroid hormone. In hypothyroid fibromyalgia, failed transcription regulation would result from thyroid-hormone deficiency. In euthyroid fibromyalgia, failed transcription regulation may result from low-affinity thyroid hormone receptors coded by a mutated c-erbAβ₁ gene, yielding partial peripheral resistance to thyroid hormone. The hypothesis of this paper is that, in euthyroid fibromyalgia, a mutant c-erbAβ₁ gene (or alternately, the c-erbAα₁ gene) results in low-affinity thyroid-hormone receptors that prevent normal thyroid hormone regulation of transcription. As in hypothyroidism, this would cause a shift toward a-adrenergic dominance and increases in both cyclic adenosine 3′-5′-phosphate phosphodiesterase and inhibitory G₁ proteins. The result would be tissue-specific hypothyroid-like symptoms despite normal circulating thyroid-hormone levels.