Regulation of basal expression of catecholamine-synthesizing enzyme genes by PACAP.

We have previously reported that the cAMP/protein kinase A (PKA) pathway is important in the gene regulation of both induction and basal expressions of the catecholamine synthesizing enzymes tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). The neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) has been shown to activate the intracellular cAMP/PKA pathway. In the present study, using primary cultured bovine adrenal medullary cells, we determined whether the basal activity of the PACAP receptor might play a role in the maintenance of the basal expression of these enzyme genes via the cAMP/PKA pathway. The potent PACAP receptor antagonist PACAP (6-38) caused a reduction of TH and DBH mRNA levels in a dose dependent manner as well as their enzyme activities and TH protein level. The effects of PACAP (6-38) and the PKA inhibitor H-89 exhibited generally similar trends, and were not additive in the reduction of TH and DBH gene expression and activities, suggesting that they take a common intracellular signaling pathway. The antagonist also caused decreases in the intracellular norepinephrine and epinephrine levels similar to the effect of H-89. Taken together, the data suggests that PACAP is involved in the regulation of maintenance of the catecholamine synthesizing enzymes TH and DBH by utilizing the cAMP/PKA pathway.     

Induction of Gene Expression of the Catecholamine-Synthesizing Enzymes by Insulin-Like Growth Factor-I

Abstract: The effects of insulin-like growth factor-I (IGF-I) on gene expression and the activities of the three enzymes specific for catecholamine biosynthesis, tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH), and phenylethanolamine N -methyltransferase (PNMT), were determined in bovine adrenomedullary chromaffin cells primary cultured in serum-free medium. The mRNA level of TH was maximally elevated in the presence of IGF-I by 3.1 ± 0.4-fold after 48 h, DBH by 5.1 ± 0.3-fold in 24 h, and PNMT by 2.8 ± 0.5-fold in 72 h. In addition, the activity of TH was increased by 77%, DBH by 70%, and PNMT by 23% in IGF-I-exposed cultures. In the absence of the growth factor, the mRNA levels of TH and DBH were decreased to 45 ± 10% and 35 ± 12% of the time-zero control within 48 h while PNMT mRNA was decreased to 82 ± 5% only after 72 h. When the cells were cotreated with the protein tyrosine kinase inhibitor genistein, DBH induction by IGF-I was suppressed, confirming that the effect is mediated by tyrosine kinase. Cotreatment with the protein kinase A (PKA) inhibitor H89 caused complete reversal of the IGF-I-induced DBH increase and the effects of IGF-I treatment and PKA activation by forskolin were not additive, suggesting that PKA is involved in the signaling initiated by IGF-I in these cells.     

Irregular electrical activation of intrinsic cardiac adrenergic cells increases catecholamine-synthesizing enzymes

Background

Recently, increased cardiac norepinephrine levels were observed in patients who were exposed to irregular stimulation during electrophysiological testing. The molecular mechanisms remain unclear. Intrinsic cardiac adrenergic (ICA) cells are present in mammalian hearts and contain catecholamine-synthesizing enzymes sufficient to produce biologically active norepinephrine levels. Thus, we aimed to investigate the expression of catecholamine-synthesizing enzymes by ICA cells exposed to irregular pacing.

Methods

Co-cultures of cardiomyocytes and ICA cells were exposed to irregular pacing for 48 h (standard deviation (SD) = 5%, 25% and 50% of mean cycle length) at a constant rate of 5 Hz. The expression of catecholamine-synthesizing enzymes including tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DBH) were analyzed on mRNA and protein levels.

Results

First, immunolabeling identified ICA cells presenting TH and DBH staining around the cell nucleus. Irregular pacing with 25% SD at a constant rate of 5 Hz significantly increased the expression of TH and DBH enzyme synthesis. Pharmacological approaches have shown that both metoprolol and losartan reversed the irregular pacing induced DBH increase, whereas the expression of TH was only blocked by metoprolol in a significant manner. Blockade of the endothelin-A receptor by BQ123 or the calcineurin–NFAT pathway by cyclosporine-A, 11R-VIVIT or FK506 revealed a potential role of both cascades in irregular pacing induced catecholamine-synthesizing enzyme expression.

Conclusions

ICA cells respond to irregular electrical activation with an increase in catecholamine-synthesizing enzymes. Drugs commonly used in clinical routine significantly influence the expression of TH and DBH by ICA cells via different signaling routes.     

Expression of mRNA for PACAP and its receptors in intra- and extra-adrenal human pheochromocytomas and their relationship to catecholamine synthesis

Purpose: Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the secretin/glucagons/vasoactive intestinal peptide family, induces the expression of catecholamine-synthesizing enzymes in adrenal medullary cells. In addition, PACAP and its receptor have been detected in human pheochromocytoma tissues, though it is not yet known whether PACAP enhances the expression of genes encoding catecholamine-synthesizing enzymes. To address this question, we analyzed PACAP, PACAP receptor, and tyrosine hydroxylase (TH) and phenylethanolamine-N-methyltransferase (PNMT) mRNAs in pheochromocytomas. Methods: The levels of the mRNA for PACAP and vasoactive intestinal peptide (VIP), and their receptors, and for TH and PNMT were measured by RT-PCR or real-time PCR analysis, and the concentrations of catecholamines were measured by HPLC in 24 intra-adrenal and six extra-adrenal pheochromocytomas. Results: mRNA expression of PACAP and its receptor VPAC1R were detected in many pheochromocytomas (24/30 and 29/30, respectively), but mRNA expression of the PAC1R and VPAC2R receptor subtypes were detected in only one of six extra-adrenal pheochromocytomas. PACAP mRNA expression correlated with TH (p=0.0018) and PNMT (p=0.05) mRNA expression, as well as epinephrine (p=0.0342) levels in 16 intra-adrenal pheochromocytomas. Conclusion: Our findings support a possible role for PACAP in the regulation of expression of genes encoding catecholamine-synthesizing enzymes in intra-adrenal pheochromocytomas.     

Immunohistochemical localization of the catecholamine-synthesizing enzymes,substance P and enkephalin in the human fetal sympathetic ganglion

Summary The human fetal sympathetic ganglia were studied using the indirect peroxidase-antiperoxidase PAP method for immunocytochemical demonstration of three catecholamine-synthesizing enzymes, tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) as well as the neuropeptides leucine (Leu⁵)-enkephalin and substance P. The neuroblasts of the ganglia showed intense peroxidase immunoreactivity for TH, moderate reaction to DBH, and no reaction to PNMT. The small intensely fluorescent (SIF) cells situated along the blood vessels also showed positive labelling for only two enzymes, TH and DBH. The immunocytochemical localization of these enzymes suggests that both neuroblasts and SIF cells synthesize noradrenalin. Neither the neuroblasts nor SIF cells showed a reaction to substance P, and only the SIF cells contained enkephalin-like immunoreactivity. The role of enkephalin in the noradrenalin-containing SIF cells is unknown, but may be related to neuromodulation of ganglionic transmission.     

Regulation of Phenylethanolamine N-Methyltransferase Gene Expression by Imidazoline Receptors in Adrenal Chromaffin Cells

Abstract: As adrenal medullary chromaffin cells express imidazoline binding sites in the absence of α₂-adrenergic receptors, these cells provide an ideal system in which to determine whether imidazolines can influence catecholamine gene expression through nonadrenergic receptors. This study evaluates the ability of clonidine and related drugs to regulate expression of the gene for the epinephrine-synthesizing enzyme phenylethanolamine N -methyltransferase (PNMT) in the rat adrenal gland and in bovine adrenal chromaffin cell cultures. In vivo, PNMT and tyrosine hydroxylase (TH) mRNA levels increase in rat adrenal medulla after a single injection of clonidine. Clonidine also dose-dependently stimulates PNMT mRNA expression in vitro in primary cultures of bovine chromaffin cells, with a threshold dose of 0.1 μ M . Other putative imidazoline receptor agonists, including cimetidine, rilmenidine, and imidazole-4-acetic acid, likewise enhance PNMT mRNA production showing relative potencies that correlate with their binding affinities at chromaffin cell I₁-imidazoline binding sites. The effects of clonidine on PNMT mRNA appear to be distinct from and additive with those exerted by nicotine. Moreover, neither nicotinic antagonists nor calcium channel blockers, which attenuate nicotine's influence on PNMT mRNA production, diminish clonidine's effects on PNMT mRNA. Although 100 μ M clonidine diminishes nicotine-stimulated release of epinephrine and norepinephrine in chromaffin cells, this effect appears unrelated to stimulation of imidazoline receptor subtypes. This is the first report to link imidazoline receptors to neurotransmitter gene expression.     

Differential involvement of PKA and PKC in regulation of catecholamine enzyme genes by PACAP.

Roles of protein kinase A (PKA) and protein kinase C (PKC) in regulation of tyrosine hydroxylase, dopamine beta-hydroxylase, and phenylethanolamine N-methyltransferase expression by pituitary adenylate cyclase-activating polypeptide (PACAP) were determined in primary cultured bovine chromaffin cells. DBH up-regulation by PACAP was reduced by H-89 and not further increased by forskolin showing involvement of cAMP/PKA. It was not mediated by PKC, as 12-O-tetradecanoylphorbol-13-acetate and sphingosine exerted no effect. Tyrosine hydroxylase induction by PACAP was mediated by both kinases. The PACAP-activated PKA up-regulated phenylethanolamine N-methyltransferase expression whereas PKC caused down-regulation. PACAP increased tyrosine hydroxylase and dopamine beta-hydroxylase activities, but slightly lowered phenylethanolamine N-methyltransferase activity, resulting in a preferential rise in norepinephrine over epinephrine.     

Long-term hypoxia modulates expression of key genes regulating adrenomedullary function in the late gestation ovine fetus

We previously communicated that long-term hypoxia (LTH) resulted in a selective reduction in plasma epinephrine following acute stress in fetal sheep. The present study tested the hypothesis that LTH selectively reduces adrenomedullary expression of phenylethanolamine-N-methyltransferase (PNMT), the rate-limiting enzyme for epinephrine synthesis. We also examined the effect of LTH on adrenomedullary nicotinic, muscarinic, and glucocorticoid receptor (GR) expression. Ewes were maintained at high altitude (3,820 m) from 30 to 138 days gestation (dGA); adrenomedullary tissue was collected from LTH and age-matched, normoxic control fetuses at 139-141 dGA. Contrary to our hypothesis, in addition to PNMT, adrenomedullary expression (mRNA, protein) of tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) were reduced in the LTH fetus. Immunocytochemistry indicated that TH and DBH expression was lower throughout the medulla, while PNMT appeared to reflect a reduction in PNMT-expressing cells. Nicotinic receptor alpha 1, 2, 3, 5, 6, 7, beta 1, 2, and 4 subunits were expressed in the medulla of LTH and control fetuses. Messenger RNA for alpha 1 and 7 and beta 1 and 2 subunits was lower in LTH fetuses. Muscarinic receptors M1, M2, and M3 as well as the GR were also expressed, and no differences were noted between groups. In summary, LTH in fetal sheep has a profound effect on expression of key enzymes mediating adrenomedullary catecholamine synthesis. Further, LTH impacts nicotinic receptor subunit expression potentially altering cholinergic neurotransmission within the medulla. These findings have important implications regarding fetal cardiovascular and metabolic responses to stress in the LTH fetus.     

Developmental profiles of catecholaminergic enzymes in adrenals of perinatal rats: effect of a hypoxic environment

Fetal and early neonatal development of adrenal catecholaminergic enzymes was studied in rats maintained under normal (normoxic) and high-altitude, 3800 m, 13% PO2 (hypoxic) conditions. In adrenals of normoxic fetuses, tyrosinehydroxylase (TH), DOPA-decarboxylase (DDC), phenylethanolamine-N-methyltransferase (PNMT), catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO) showed rapid increases in activity from day 19 to day 21 of gestation. The activities of all enzymes but TH were higher at day 1 postpartum compared to fetal values: TH was equiactive just before and after birth. In animals conceived, born and raised at high altitude, several changes indicative of impaired adrenal development occurred. The activities of the synthesizing enzymes, TH, DDC and PNMT, were variably affected at some time during the perinatal period. The activities of the catabolizing enzymes, MAO and COMT, at high altitude were increased on the last days of gestation but depressed after birth, compared to control levels. Catecholamine content in high-altitude adrenals was altered on day 19 of gestation when epinephrine was lower, and again on day 1 postpartum when both norepinephrine and epinephrine were higher than in control adrenals at sea level. Normal developmental changes and high-altitude-induced disturbances in adrenal catecholaminergic enzymes are discussed with reference to differences observed in adrenal cortical function between sea-level and high-altitude animals.     

Subcellular Site of Biosynthesis of the Catecholamine Biosynthetic Enzymes in Bovine Adrenal Medulla

The subcellular site of biosynthesis of the catecholamine biosynthetic enzymes was examined. Free and membrane-bound polysomes were prepared from bovine adrenal medulla and mRNA was isolated from these polysomes. Both were active in directing cell-free translations. Immunoprecipitation of cell-free products with specific antisera localized the biosynthesis of the subunits of tyrosine hydroxylase (TH) (apparent Mr = 61,000) and of phenylethanolamine N-methyltransferase (PNMT) (apparent Mr = 32,000) on free polysomes, compared with biosynthesis of subunits of dopamine beta-hydroxylase (DBH) (apparent Mr = 67,000) on membrane-bound polysomes. Cross-reactivity between translation products was observed. Antibodies for DBH recognized a polypeptide with electrophoretic mobility identical to newly synthesized PNMT. However increasing concentrations of antibodies to DBH recognized at most 1/20 of the PNMT formed. The results of this study show the subcellular distribution of the catecholamine synthesizing enzymes is determined by their site of biosynthesis.     

Short and long term regulation of catecholamine biosynthetic enzymes by angiotensin in cultured adrenal medullary cells. Molecular mechanisms and nature of second messenger systems

The purpose of this study was to examine the effects of angiotensin on the enzyme activities and gene expression of two catecholamine synthesizing enzymes, tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT), in bovine adrenal medullary (AM) cells. Short term (15 min) incubation of cultured AM cells with 2 nM [Sar1]angiotensin II (s1-AII) did not increase basal secretion of catecholamines; however, longer incubations (3, 24, or 72 h) produced 4-10-fold increases. To determine whether angiotensin affects synthesis of catecholamines, the activities of TH and PNMT were examined. Incubation with s1-AII (15-30 min) decreased the Km of TH for its biopterine cofactor [6R)-5,6,7,8-tetrahydro-1-biopterin dihydrochloride (BH4] without affecting the Vmax, suggesting activation of TH. After long term incubation (72 h) the Km value was identical to that of control, while increases in the apparent Vmax were observed. PNMT activity was unaffected during a 30-min treatment with s1-AII; however, 2-fold increases occurred after a 48-72-h incubation. s1-AII (24 h) increased the relative abundance of TH and PNMT mRNAs, suggesting that the long term increase in enzyme activities reflected increased expression of TH and PNMT genes. Maximal increases were observed at 2 nM s1-AII and the changes were antagonized by saralasin. Induction of TH mRNA by s1-AII was additive to the effects of veratridine or forskolin indicating that effects of angiotensin were not due to membrane depolarization or increased cyclic AMP levels. Incubation with Ca2+ ionophore A23187 increased TH and PNMT mRNA levels in AM cells raising the possibility that the increase in cellular [Ca2+] could mediate effects of angiotensin. Angiotensin-induced increases in TH and PNMT mRNA were inhibited by nifedipine indicating involvement of voltage-dependent Ca2+ channels. In addition, the increases in TH, but not PNMT mRNA, were antagonized by dantrolene, which inhibits mobilization of Ca2+ from intracellular stores. Calmodulin involvement was suggested by the inhibition of s1-AII induced changes in mRNA with 1 microM calmidazolium.(ABSTRACT TRUNCATED AT 400 WORDS)     

Parallel Up-Regulation of Catecholamine Biosynthetic Enzymes by Dexamethasone in PC12 Cells

Abstract: We sought to investigate whether dexamethasone produces a coordinated, time-dependent effect on all enzymes in the catecholamine biosynthetic pathway in PC12 cells. The levels of mRNAs of tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), and dopamine γ-hydroxylase (DBH) were examined at 0, 6, 12, 24, and 48 h after dexamethasone (5 μ M ) treatment to PC12 cells. The levels of all enzyme mRNAs steadily increased for 24 h, although the increase of AADC mRNA content was slow. The increased mRNA levels of TH and AADC were maintained at 48 h, whereas the level of DBH mRNA was sharply decreased at 48 h. The maximally induced mRNA levels were ∼5.0-, 2.4-, and 7.0-fold higher than the control levels of TH, AADC, and DBH, respectively. The elevation of enzyme activities was detected later than the increase in levels of mRNAs. The maximal activities of TH, AADC, and DBH were reached between 48 and 72 h with 3.6-, 1.8-, and 8.0-fold increases, respectively. Low, but detectable, phenylethanolamine N -methyltransferase (PNMT) activity was observed in PC12 cells, and dexamethasone increased its activity 5.6-fold at 72 h. The PNMT mRNA was easily detected by northern blot analysis after exposure for 24 h to dexamethasone. The data suggest that, in PC12 cells, dexamethasone up-regulates all catecholamine biosynthetic enzyme genes in a parallel fashion.     

Regulation of atrial catecholamine enzymes and adrenoceptor gene expression after chronic stress

Chronic stress is a risk factor for the development of numerous psychopathological conditions in humans including depression. Changes in gene expression of tyrosine-hydroxylase (TH), dopamine-β-hydroxylase (DBH) phenylethanolamine N-methyltransferase (PNMT), β1-, β2- and β3-adrenoceptors in right and left rat atria upon chronic unpredictable mild stress (CMS) were investigated. CMS decreased TH and DBH gene expression levels both in right and left atria and increased PNMT mRNA in left atria. No changes in mRNA levels of β1- and β2-adrenoceptors were recorded, whereas β3-adrenoreceptor mRNA level was significantly elevated in right atria of CMS rats. At the same time, CMS produced a significant increase of β1- and β2-adrenoreceptor mRNA levels in left atria, but did not affect β3-adrenoceptor mRNA level.The results presented here suggest that stress-induced depression expressed differential effects on catecholamine biosynthetic enzymes and β-adrenoceptors at molecular level in right and left atria of adult rat males. Elevated gene expression of PNMT in left atria of rats exposed to CMS can lead to altered physiological response and may play a role in the pathophysiology of cardiovascular function.     

Differentiation of adrenomedullary catecholamine synthesizing enzyme responses to repeated immobilization in hybrid rats

The response of adrenomedullary catecholamine synthesizing enzymes to repeated immobilization was studied in hybrid (F₁) offspring of 2 inbred rat strains (LEW and F344). Immobilization-induced increases in tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine-N-methyl-transferase (PNMT) activities in one of the parental strains (F344) previously were shown to be dependent upon intact adrenal gland innervation but independent of the pituitary gland, while responses in the other parental strain (LEW) were independent of adrenal innervation but dependent upon pituitary function. Factors determining immobilization-induced increases in adrenal enzymes of F₁ offspring were enzyme-specific. Increased PNMT activity was pituitary dependent in F₁ rats, whereas increased TH and DBH activities after immobilization were dependent upon an intact adrenal gland innervation. These results suggest that the factor(s) regulating PNMT responses are differentiable from those regulating TH and DBH responses. The results also indicate that analysis of PNMT responses to immobilization in backcross populations is feasible, and could indicate whether strain-specific response mechanisms are heritable.     

Catecholamine Metabolism in Paraganglioma and Pheochromocytoma: Similar Tumors in Different Sites?

Pheochromocytoma (PHEO) and paraganglioma (PGL) are catecholamine-producing neuroendocrine tumors that arise respectively inside or outside the adrenal medulla. Several reports have shown that adrenal glucocorticoids (GC) play an important regulatory role on the genes encoding the main enzymes involved in catecholamine (CAT) synthesis i.e. tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). To assess the influence of tumor location on CAT metabolism, 66 tissue samples (53 PHEO, 13 PGL) and 73 plasma samples (50 PHEO, 23 PGL) were studied. Western blot and qPCR were performed for TH, DBH and PNMT expression. We found a significantly lower intra-tumoral concentration of CAT and metanephrines (MNs) in PGL along with a downregulation of TH and PNMT at both mRNA and protein level compared with PHEO. However, when PHEO were partitioned into noradrenergic (NorAd) and mixed tumors based on an intra-tumoral CAT ratio (NE/E >90%), PGL and NorAd PHEO sustained similar TH, DBH and PNMT gene and protein expression. CAT concentration and composition were also similar between NorAd PHEO and PGL, excluding the use of CAT or MNs to discriminate between PGL and PHEO on the basis of biochemical tests. We observed an increase of TH mRNA concentration without correlation with TH protein expression in primary cell culture of PHEO and PGL incubated with dexamethasone during 24 hours; no changes were monitored for PNMT and DBH at both mRNA and protein level in PHEO and PGL. Altogether, these results indicate that long term CAT synthesis is not driven by the close environment where the tumor develops and suggest that GC alone is not sufficient to regulate CAT synthesis pathway in PHEO/PGL.     

Effects of diabetes and recurrent hypoglycemia on the regulation of the sympathoadrenal system and hypothalamo-pituitary-adrenal axis

Epinephrine, norepinephrine, and corticosterone responses to hypoglycemia are impaired in diabetic rats. Recurrent hypoglycemia further diminishes epinephrine responses. This study examined the sympathoadrenal system and hypothalamo-pituitary-adrenal axis for molecular adaptations underlying these defects. Groups were normal (N) and diabetic (D) rats and diabetic rats exposed to 4 days of 2 episodes/day of hyperinsulinemic hypoglycemia (D-hypo) or hyperinsulinemic hyperglycemia (D-hyper). D-hypo and D-hyper rats differentiated effects of hypoglycemia and hyperinsulinemia. Adrenal tyrosine hydroxylase (TH) mRNA was reduced (P < 0.05 vs. N) 25% in all diabetic groups. Remarkably, mRNA for phenylethanolamine N-methyltransferase (PNMT), which converts norepinephrine to epinephrine, was reduced (P < 0.05 vs. all) 40% only in D-hypo rats. Paradoxically, dopamine beta-hydroxylase mRNA was elevated (P < 0.05 vs. D, D-hyper) in D-hypo rats. Hippocampal mineralocorticoid receptor (MR) mRNA was increased (P < 0.05 vs. N) in all diabetic groups. Hippocampal glucocorticoid receptor (GR), hypothalamic paraventricular nucleus (PVN) GR and corticotropin-releasing hormone (CRH), and pituitary GR and proopiomelanocortin (POMC) mRNA levels did not differ. We conclude that blunted corticosterone responses to hypoglycemia in diabetic rats are not due to altered basal expression of GR, CRH, and POMC in the hippocampus, PVN, and pituitary. The corticosterone defect also does not appear to be due to increased hippocampal MR, since we have reported normalized corticosterone responses in D-hypo and D-hyper rats. Furthermore, impaired epinephrine counterregulation in diabetes is associated with reduced adrenal TH mRNA, whereas the additional epinephrine defect after recurrent hypoglycemia is associated with decreases in both TH and PNMT mRNA.     

Increased gut-derived norepinephrine release in sepsis: up-regulation of intestinal tyrosine hydroxylase

Studies have shown that increased gut-derived norepinephrine (NE) release plays an important role in producing hepatocellular dysfunction at the early stage of sepsis. Although the gut has been demonstrated to be the major source of NE in sepsis, it remains unknown whether the increased NE is associated with up-regulation of intestinal NE biosynthesis enzymes such as tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). To determine this, adult male rats were subjected to sepsis by cecal ligation and puncture (CLP) followed by fluid resuscitation. Small intestinal samples were harvested at 2 h (i.e., early sepsis) or 20 h (late sepsis) after CLP or sham-operation. Protein levels of TH and DBH were determined by Western blot analysis and immunohistochemistry. Their gene expression was assessed by RT-PCR technique. The results indicate that intestinal TH protein levels increased significantly at 2 and 20 h after CLP, while DBH was not altered under such conditions. Immunohistochemical examination shows that both TH and DBH were located in intestinal sympathetic nerve fibers and TH staining was markedly increased in septic animals. TH gene expression increased significantly at 2 h but not at 20 h after CLP, while DBH gene expression was not altered in sepsis. Thus, the increased TH gene and protein expression appears to be responsible for the increased gut-derived NE in sepsis.     

Phenylethanolamine N-methyltransferase-(PNMT)-like immunoreactivity in the rat parathyroid gland

A phenylethanolamine N-methyltransferase-(PNMT)-immunoreactivity, present without the other catecholamine-synthesizing enzymes tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH), has been previously detected in the central nervous system and in endocrine cells of the islets of Langerhans and the pituitary intermediate lobe of the rat. In the present study a similar PNMT-like immunoreactivity is demonstrated in the rat parathyroid gland. The immunoreactivity was distinctly localized to the cell periphery, and present in all glandular cells. The thyroid gland was negative. In the parathyroid TH- and DBH-immunoreactivity was seen only in vascular nerve fibers; no glandular cells were stained. The functional significance of the PNMT-like immunoreactivity is not known. The absence of TH- and DBH-immunoreactivity and the low level of adrenaline detected in the parathyroid, and the peripheral localization of the immunoreactivity may indicate an alternative enzyme function or the detection of an immunologically related protein common to pancreatic, pituitary and parathyroid endocrine cells.