Copyright 0 1984 by the Genetics Society of America INHERITANCE OF ADRENAL PHENYLETHANOLAMINE N-METHYLTRANSFERASE ACTIVITY IN THE RAT JON M. STOLK,* GUIDO VANTINI,* RAS B. GUCHHAIT,* JEFFREY H. HURST,* BRUCE D. PERRY: DAVID C. U’PRICHARDt and ROBERT C. ELSTON* *Maryland Psychiatric Research Center, University of Maryland School of Medicine, Baltimore, Maryland 21228; tDepartment of Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611; and *Department of Biometry, Louisiana State University Medical Center, New Orleans, Louisiana 70112 Manuscript received February 16, 1984 Revised copy accepted June 14, 1984 ABSTRACT Phenylethanolamine N-methyltransferase (PNMT) is the enzyme that catalyzes the S-adenosyl-L-methioninedependentmethylation of (-)norepinephrine to (-)epinephrine in the adrenal medulla. Adrenal PNMT activity is markedly different in two highly inbred rat strains; enzyme activity in the F344 strain is more than fivefold greater than that in the Buf strain. Initial characterization of the enzyme in the two inbred strains reveals evidence for catalytic and structural differences, as reflected in dissimilar K, values for the cosubstrate (S-adenosyl-L- methionine) and prominent differences in thermal inactivation curves. To assess adrenal PNMT activity in an F344 X Buf pedigree, we employed a statistical procedure to test for one- and two-locus hypotheses in the presence of within- class correlations due to cage or litter effects. The PNMT data in the pedigree are best accounted for by segregation at a simple major locus superimposed upon a polygenic background; data obtained from the biochemical studies suggest that the major locus is a structural gene locus. HENY LETHANOLAMINE N-methyltransferase (PNMT) is the enzyme that p catalyzes the S-adenosyl-L-methionine (SAMe)-dependent methylation of norepinephrine to epinephrine (AXELROD1962). In mammals, the highest con- centrations of PNMT are found in the adrenal medulla; lower levels of enzyme activity have been observed in other tissues, including sympathetic ganglia (MOOREand PHILLIPSON1975) and the brain (HOKFELTet al., 1974; SAAVEDRA et al., 1974). As the enzyme responsible for epinephrine biosynthesis, PNMT has been attributed with an important role in homeostasis, and the regulation of its activity in the adrenal medulla has been extensively evaluated. Primary physio- logical influences on adrenomedullary PNMT regulation are well documented and include (1) glucocorticoids (WURTMANand AXELROD1966), (2) adrenocor- ticotropin, acting either directly on adrenomedullary PNMT-containing cells (MUELLER,THOENEN and AXELROD1970) or acting indirectly through its effects on the adrenal cortex and (3) innervation of the adrenal medulla via the splanchnic bed (KVETNANSKY,WEISE and KOPIN 1970). The mechanisms that regulate adrenomedullary PNMT in response to environmental and physiological Genetics 108: 633-649 November, 1984 634 J. M. STOLK ET AL. challenge have been documented to be strain specific in both mice (CIARANELLO, DORNBUSCHand BARCHAS1972) and rats (COOPERand STOLK1979); evidence suggesting inheritance of specific mechanisms for regulating the response of adrenal PNMT activity in stress has been presented (STOLKet al. 1980; STOLK and HARRIS1980). Inheritance of basal adrenomedullary PNMT levels has been studied in inbred mouse strains (KESSLER et al. 1972) and in sublines of the BALB/cJ strain (CIARANELLOand AXELROD1973). Adrenal PNMT levels in the BALB/cJ sublines were reported to be inherited as an autosomal codominant trait trans- mitted at a single gene locus (CIARANELLOand AXELROD1973). Although isozymes of PNMT have been suggested by disc gel electrophoresis (JOH and GOLDSTEIN1973) and isoelectric focussing (LEE,SCHULZ and FULLER1978a-c), no evidence for structural differences was found in the BALB sublines by CIARANELLOand AXELROD(1 973). Rather, differences between the sublines in the rates of PNMT degradation accounted for the different steady-state adrenal enzyme levels. Subsequent evaluation of all three enzymes responsible for cate- cholamine biosynthesis (tyrosine hydroxylase, dopamine j3-hydroxylase and PNMT) suggested that differences in steady-state levels of each enzyme in the BALB sublines could be accounted for by a common mechanism-altered enzyme protein degradation (CIARANELLOet al. 1974). Based upon their statistical analysis of data for the three enzymes, CIARANELLOet al. (1974) concluded that a single autosomal codominant gene locus was responsible for the differences in all three catecholamine synthetic enzymes in the adrenal glands of the BALB/cJ sublines; they hypothesized that adrenomedullary enzyme levels were under the control of a regulatory gene, expressed through the rates of enzyme protein degradation. The data of CIARANELLOet al. (1974) subsequently were reevalua- ted by ELSTON (1981) using a new nonparametric method to test whether segregation at a single locus can account for the variability of adrenal PNMT activity in the BALB/cJ sublines. Using the latter method, ELSTON (198 1) concluded that the mechanism of inheritance of adrenal PNMT in the BALB/cJ sublines studied by CIARANELLOet al. (1974) is more complicated than simply single-locus control. Recent observations in our laboratory suggested that the conclusions reached by CIARANELLOet al. (1974) in the mouse did not appear to generalize to the rat (STOLKet al. 1980; STOLKand HARRIS1980). Specifically, analysis of tyrosine hydroxlyase, dopamine P-hydroxylase and PNMT activities in the adrenal glands of inbred and hybridized rat strains could not support a common inherited regulatory mechanism. During the course of our subsequent investigations into the mechanisms regulating adrenomedullary catecholamine synthesizing en- zymes, an inbred rat strain (Buffalo strain: Buf) with remarkably low adreno- medullary PNMT was encountered. The fivefold difference between adrenal PNMT activity in the Buf strain and other highly inbred strains in our colony prompted reevaluation of the mechanisms of inheritance of basal adrenal enzyme levels in the rat. Incorporated into our assessment of adrenomedullary PNMT inheritance is the application of a statistical procedure to test for one- and two- locus hypotheses in the presence of within-class correlations due to cage and/or litter effects (ELSTON1984). RAT ADRENAL PNMT INHERITANCE 635 MATERIALS AND METHODS Animals Experimental subjects were derived from colonies of F344 and Buf inbred rat strains bred in our facility by strict brother-sister mating. The colonies came initially from Microbiological Associates (Walkerville, Maryland) parental stock inbred by brother-sister mating for at least 100 (F344) or 65 (Buf) generations when transferred to our facility. All rats in our colony are housed with littermates of the same sex to a maximum of six rats per cage; rats have free access to food and water at all times. Lights in the animal rooms were on from 0600 to 1800 hr. Standard breeding procedures were to introduce one male rat into a group of from two to five female littermates for a 10day period; bred females were isolated at the end of the 10-day period. No attempt was made to cull litters to a maximal size, and all litters were weaned from their biological mothers at 29 days of age. Biochemical measures were performed on adrenal gland tissue obtained at sacrifice (guillotine) when offspring were between 84 and 98 days old. Biochemical measures Tissue preparation: Adrenal glands were removed rapidly after decapitation, dissected free of fat and either frozen on dry ice (pedigree study) or processed immediately for enzyme assay (biochemical characterization of PNMT). PNMT assayfor the pedigree study: Adrenal glands were homogenized in 4.0 ml of 10 mM Tris-HCI buffer (pH 7.4) containing 0.1 % (v/v) Triton X-100 with a Polytron (Brinkmann Instruments; 5 sec, setting no. 7) and centrifuged at 15,000 X g for 15 min. PNMT activity was measured in the supernatant fraction by the procedure described initially by AXELROD(1962). The PNMT substrate was phenylethanolamine (final concentration, 1 mM), and the methyl donor was ['4C-methyl]-SAMe (56.2 mCi/mmol; New England Nuclear). Enzyme activity is expressed as units per adrenal gland pair, where 1 unit of PNMT activity is defined as the formation of 1 X lo-' mol of product/hr of incubation at 37". Boiled adrenal supernatant served as the reaction blank; PNMT activity for each subject was determined from duplicate aliquots of adrenal supernatant. The intraassay coefficient of variance is less than 2%, and the interassay coefficient of variance is less than 4%, for the assay procedure. The PNMT activity values in the pedigree study were obtained from six independent assays conducted over 3 wk using identical reagents. PNMT kinetic parameters: Fresh adrenal glands were homogenized in 4.0 ml of 10 mM Tris-HCI buffer (pH 8.6) and centrifuged as described. Endogenous catecholamines were removed by adsorp tion to alumina. The catecholamine-free supernatants were used immediately for kinetic studies. (-)Norepinephrine was the PNMT substrate in all kinetic studies. The formation of epinephrine from supernatants incubated at 37 O with known concentrations of substrate and SAMe was measured by high pressure liquid chromatography (HPLC). Briefly, enzyme reactions were terminated by addition of perchloric acid, and catecholamines were adsorbed to alumina at pH 8.6; the alumina (with adsorbed catechols) was washed three times with 5 mM Tris-HCI buffer (pH 8.6), and the catecholamines