Inheritance of Adrenal Phenylethanolamine N-Methyltransferase Activity in the Rat

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 concentrations 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 physiological 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 catecholamine 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 enzymes, an inbred rat strain (Buffalo strain: Buf) with remarkably low adrenomedullary 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 were eluted with 0.4 N perchloric acid. Epinephrine concentration was determined amperometrically using a Bioanalytic Systems system (model LC-3 detector and glassy carbon electrode) after chromatography on a 25-cm reversed-phase C-18 column (5 pm, Bioanalytical Systems) with a mobile phase of 0.1 M phosphate buffer (pH 3) containing sodium octyl sulfate (5 mg/liter), 1 mM EDTA and 5% (v/v) methanol. Dihydroxybenzylamine served as an internal standard in all samples (added with the perchloric acid to stop the PNMT assay), and product formation was determined by integration of the amperometric signal using a Hewlett-Packard model 3390A integrator. Determination of the K, for (-)norepinephrine was calculated from double reciprocal plots (by least squares regression analysis) in the presence of 20 p~ SAMe. Determination of the SAMe K, was calculated by the same procedure in the presence of 40 p~ (-)norepinephrine. PNMT thermolability: Fresh adrenal supernatants were prepared as described for the PNMT kinetic studies. Aliquots (240 PI) from the supernatant of a given rat were incubated at 50.5" for timed intervals before being placed in an ice water bath. PNMT activity was measured using the radiochem- ical assay described in the preceding section. The decrement in PNMT activity with time of incubation at 50.5" was measured in duplicate samples. 636 J. M. STOLK ET AL.

Adrenal catecholamine concentration: Norepinephrine and epinephrine concentrations of F344 and Buf rats were determined in aliquots of adrenal gland homogenates precipitated with perchloric acid containing a known concentration of the internal standard, dihydroxybenzylamine. Adsorption of catecholamines to alumina and determination by HPLC followed the procedure described earlier. Adrenal tyrosine hydroxylase activity: Tyrosine hydroxylase activity was determined in adrenal gland honiogenates of an independent group of F344 and Buf male rats using the procedure of WAYMIRE, BJUR and WEINER(1 97 1). Enzyme activity is expressed as units per adrenal gland pair, where 1 unit is defined as the formation of 1 X lo-’ mol of COn/hr of incubation at 30”. Adrenal dopamine &hydroxylase activity: Dopamine &hydroxylase activity was determined in the same subjects used for tyrosine hydroxylase measurements; enzyme activity was estimated by the procedure of MOLINOFF,WEINSHILBOUM and AXELROD(1 97 1) using tyramine as the enzyme substrate. Endogenous inhibitors of dopamine @hydroxylase were neutralized by cupric chloride (final concentration, 10 PM). One unit of enzyme activity is defined as the formation of 1 X IO-’ mol of N- methyl-octopamine/hr of incubation at 37 O. Statistical analyses Adrenal PNMT activity values in the entire pedigree were subjected to the methods of analysis discussed by ELSTON(1984), which should be consulted for statistical details. Initial inspection of the data indicated that, although the reciprocal backcrosses could be pooled, there are marked sex differences; therefore, the two sexes were analyzed separately. Initially, a power transformation of the data was determined by maximum likelihood from the parental and FI rats to approximate homoscedasticity and normality. Then, with the use of that power transformation, maximum likelihood methods were used to fit genetic hypotheses on the assumption that all genotypes give rise to normal phenotypic distributions with two components of environmental variance-a cage component (with which any litter effect is confounded) and a random component. Thus, the cage component of variance includes any variation due to differences among litters and is identified with the litter component of variance in the models described by ELSTON(1984). The following genetic hypotheses are considered. One locus: The general one-locus hypothesis (A-I: notation refers to that followed in Table 4, RESULTS) allows the FI mean to be arbitrary; if the genes are additive (A-2), the Fl mean is half-way between the parental means. Polygenic: The general (B-1) and additive genes (B-2) polygenic hypotheses are analogous but allow for the segregation of a large number of equal and additive loci. As the number of such loci tends to infinity, the backcross variance becomes the same as the variance within the parental strains and we obtain, correspondingly, the hypotheses of infinite polygenic general (B-3) and infinite polygenic additive genes (B-4). Mixed: The general mixed hypothesis (C-1) assumes both a major locus and a polygenic component; these two components act additively with each other. The major locus may have additive genes (C- 2), and this may be combined with additivity at the polygenic loci to give complete additivity (C-7). Without assuming any additivity (other than that between the major locus and the polygenic components), it is possible to apply one of two mild symmetry restrictions (ELSTON 1984; ELSTON and STEWART1973) that relate the component means in the two backcross distributions to the means of their respective parents: symmetry A (C-3) and symmetry B (C-4). Each of these symmetry conditions may also be combined with additivity at the major locus (C-5, C-6); complete additivity implies both of them. Two loci: Each two-locus hypothesis allows for the loci to be either unlinked (recombination fraction = 0.5) or linked (recombination fraction arbitrary). In the most general case (D-1, D-10) the phenotypic distributions for the nine possible genotypes can have arbitrary means. There may be additivity between the two loci (D-2, D-1 I), symmetry A (D-3, D-12) or symmetry B (D-4, D-13); in this case the symmetry conditions relate the means of the recombinant distributions to the means of the nonrecombinant distributions. If there is additivity between the two loci and the F1 mean lies half-way between the parental means, we have additive loci with zero average dominance (D-5, D- 14). Additivity between the two loci with symmetry A implies that the two loci have equal dominance ratios when the loci are in coupling (D-6, D-l5), whereas with symmetry B it implies equal dominance ratios when they are in repulsion (D-7, D-16). Alternatively, we may assume two loci whose effects RAT ADRENAL PNMT INHERITANCE 637 are both equal and additive (D-8, D-17), and, finally, this may be combined with additivity of the genes within each locus (D-9, D-18). The hypothesis that best fits the data is determined on the basis of AKAIKE’Smaximum entropy criterion (AKAIKE1977); this is essentially the same as choosing the hypothesis that maximizes the likelihood of the data but making an adjustment for the fact that the number of parameters estimated from the data varies from one hypothesis to another. In addition, the fit of each hypothesis is determined by the use of a chi-square statistic; this measures the significance of the departure from an overall model that6ubsumes all of the hypotheses examined. Because of the excessive amount of computation that would have been required, it was not possible to perform an appropriate analysis that included the F2 data. Analyses that included the F2 were, however, performed by the expedient of splitting cages containing more than three rats into two, i.e., by assuming the rats were grouped into two separate cages. The results of such analyses were compared with those of analogous analyses that assumed no cage component of variance (i.e., only one rat per cage), both including and excluding the FP data.

RESULTS Adrenomedullary biochemical measures in parental strains: Catecholamine biosynthetic enzyme activity and catecholamine concentration in male F344 and Buf rats are summarized in Table 1. There are significant differences in tyrosine hydroxylase activity and norepinephrine content, with Buf male rats having higher levels than F344 rats for both measures; F344 rats, however, have higher dopamine /3-hydroxylase activity than Buf rats. The major between-strains difference was in PNMT activity, with F344 rats having about fivefold higher enzyme activity than Buf rats (Table 1). Paradoxically, Buf rats had slightly, but significantly, higher adrenal gland epinephrine content than F344 rats, despite having much lower levels of the enzyme responsible for epinephrine biosynthesis. The large differences in adrenomedullary PNMT activity prompted an initial characterization of kinetic and physical properties of the enzyme in the two parental strains. The K, for the endogenous PNMT substrate, (-)norepinephrine, was the same in F344 and Buf rats (Table 2); as suggested by the data in Table 1, the Vmavfor F344 rats was more than fivefold greater than that for Buf rats (Table 2). Similar assessments were carried out for SAMe, the PNMT cosubstrate. In contrast to the similarities in the norepinephrine K, values, there was a significant difference in the K, for SAMe in the enzyme from F344 and Buf rats. The SAMe K, in F344 rats was twofold higher than that in Buf rats (Table 2). These data suggest that there may be physical differences in PNMT from F344 and Buf rats that are manifest in different affinities for SAMe but not for norepinephrine. The possible existence of PNMT structural variants in the two rat strains was assessed in a preliminary manner by evaluating the thermolability of the enzyme in adrenal supernatants. Relative enzyme thermolability remains one of the most sensitive and accurate measures of differences in protein structure (PAIGEN197 1) and has been used previously to assess mouse and rat adrenomedullary PNMT (CIARANELLOand AXELROD1973; CIARANELLO,WONG and BERENBEIM1978). Representative data for PNMT thermolability in fresh F344 and Buf adrenal supernatants are illustrated in Figure 1. Thermal denaturation of PNMT in freshly prepared adrenal supernatants of F344 rats is qualitatively distinct from that of Buf rats. Denaturation of PNMT in F344 adrenal supernatants is relatively 638 J. M. STOLK ET AL.

TABLE 1

Biosynthetic enzyme activity and catecholamine concentration (*SO) in F344 and Buj inbred rat strains

Measure F344 (N = 5) Buf (N = 5)” Biosynthetic enzymes (units/adrenal pair) Tyrosine hydroxylase 18.3 (2.9) 25.4 (1.1) Dopamine &hydroxylase 264 (36) 216 (10) PNMT 6.18 (0.26) 1.35 (0.07) Catecholamines (nmol/adrenal pair) Norepinephrine 28.0 (1.8) 65.8 (7.1) Epinephrine 146 (2) 166 (17) Each is significant (P < 0.05) compared with value for F344 rats.

TABLE 2

Kinetic parameters (~sD)of adrenomedullary PNMT in F344 and Buf inbred rat strains

F344 (N = 3) Buf (N = 5) Norepinephrine Km (PM) 12.3 (3.8) 14.8 (2.7) V,, (units/adrenal) 37.2 (8.3) 8.3 (1.4)* S-Adenosy l-L-methionine Km (W) 5.97 (0.45) 2.83 (0.23)* * Denotes a significant difference (P < 0.05) compared with value for F344 rats. slow and does not follow first-order kinetics, whereas that in Buf adrenal supernatants is rapid and apparently first order (Figure 1; approximate t1I2 = 2.4 min). Dilution of the F344 supernatant does not modify the differences in enzyme thermolability in the two inbred rat strains. Inheritance of adrenomedullary PNMT activity in the F344 X Buf pedigree: Raw values for adrenomedullary PNMT activity in the pedigree are summarized in Table 3. The average litter size is 4.7 males and 5.3 females. There are marked sex differences but no significant differences between reciprocal F1 or backcross offspring. Analysis of the parental and F1 classes indicated that after an appropriate power transformation of PNMT activity there is no significant heteroge- neity among either the cage or random components of variance in the three classes (x2with 4 d.f. = 1.23 for males and 4.06 for females) and that the cage component is highly significant (x2with 1 d.f. = 33.45 for males and 21.59 for females). Essentially the same result was obtained if cages containing more than three individuals were split in two, although the cage component then, as was to be expected, is less significant (x2 with 1 d.f. = 18.72 for males and 9.85 for females). In either case the appropriate power transformations were found to be approximately x = yo.2for males and x = for females, where y is the measured PNMT activity; these transformations were, therefore, used for all subsequent analyses. It was noted that when the estimated class means was transformed back to the original scale the three female means were approximately a constant RAT ADRENAL PNMT INHERITANCE 63 9

10 ' ' I I 1 024 8 16 24 Minutes atSQSC FIGURE1 .-Thermal inactivation curves for PNMT activity in fresh adrenal gland homogenates from F344 (circles) and Buf inbred rat strains (squares). Identical aliquots of each homogenate were incubated at 50.5" for different time periods prior to measurements of enzymatic activity (see MATERIALS AND MTHODS for details). PNMT activity within each strain is expressed as a percentage of the initial activity (0 min incubation at 50.5") and is plotted on a log scale us. minutes of heat treatment.

TABLE 3

Adrenomedullary PNMT activity in an F344 X Buf pedigree

Male offspring Female offspring

Population Litters N Mean (sD) Litters N Mean (SD) F344 10 41 6.53 (0.47) 5 32 7.97 (0.34) Buf 11 34 1.34 (0.12) 8 26 1.59 (0.17) FI Pooled 11 43 3.80 (0.27) 8 38 4.95 (0.28) F344 X Buf 5 21 3.88 (0.28) 4 20 4.99 (0.26) Buf X F344 6 22 3.73 (0.23) 4 18 4.90 (0.30) Fz 19 114 3.57 (1.55) 19 103 4.49 (1.77) FI X F344 (pooled) 15 76 4.94 (1.05) 11 61 6.36 (1.36) FI X Buf (pooled) 12 61 2.95 (1.19) 11 68 2.95 (1.43) 640 J. M. STOLK ET AL. multiple (1.2) of the male means. This suggests that the same basic genetic mechanism may be underlying PNMT activity in both sexes, the different transformation required for homoscedasticity indicating a sex-specific genotype- environment interaction. The results of the genetic analysis of the transformed PNMT values, excluding the F2 data, are summarized in Table 4. For males, the hypothesis that fits the data best (as judged by the maximum entropy criterion being minimal) is the mixed major locus/polygenic hypothesis with symmetry B (C-4); the only hypotheses that fit the data (in the sense that they do not give rise to chi-square values significant at 5%) are the general mixed hypothesis (C-1) and this same hypothesis with either of the two symmetry conditions (C-3, C-4). For females, the best hypothesis is again the mixed one, but with symmetry A (C-3); the only hypotheses that do not lead to chi-square values significant at the 5% level are the mixed ones (C-1, C-3, C-4) and the general two-locus hypotheses (D-1, D- 1O). Figures 2 and 3 display the backcross data graphically as empirical cumulative plots: the rank of each individual subject’s PNMT activity, divided by the number of rats in the class plus 1, is plotted (ordinate) against PNMT activity (abscissa); 1 is the rank of the smallest value and n the rank of the largest value. In each case the distribution of the backcross data is compared with the theoretical distribution predicted by fitting the mixed major locus/polygenic hypothesis with a symmetry condition. The plots for the Buf backcross (Figures 2B and 3B) indicate a small discrepancy in the numbers expected in the component distributions: there is a deficiency of males, but an excess of females, in the lower distribution compared with the number expected. Within the component distributions, however, the fit is good, as is the overall fit of the F344 backcross in both sexes (Figures 2A and 3A). The fit of the parental and F1 data to the theoretically estimated distributions is displayed in Figures 4 and 5. In these figures the Buf data have been plotted in the bottom quarter of the graph, the F, data in the middle half and the F344 data in the top quarter. Thus, in addition to illustrating how well the theoretical distributions fit the parental and F1 data, these plots indicate the F2 distributions that should be observed under simple monogenic segregation. When the F2 rats are included in the analysis (the cages containing more than three rats are split into two cages), the data apparently no longer fit the mixed major gene/polygenic hypothesis. This, however, may be a reflection of the fact that it was necessary, for computational reasons, to split the litters and, hence, assume a certain amount of nonexistent independence; the fit was even worse when complete independence (i.e., one rat per cage) was assumed. Nevertheless, the maximum likelihood estimates of the component means obtained under this hypothesis are very similar whether the F2 data are excluded or included in the analysis; the maximum discrepancy is 0.01 for males and 0.02 for females. Figures 6 and 7 show empirical cumulative plots of the F2 data, together with the theoretical distributions predicted from the non-F2 data. In both sexes there is a small excess of individuals in the middle component distribution, but otherwise the fit is good. If the distributions in Figures 6 and 7 are compared RAT ADRENAL PNMT INHERITANCE 64 1

TABLE 4

Akaike’s maximum entropy criteria and x2 goodness of fit statistics corresponding to alternative genetic hypotheses for PNMT activity (F2 data excluded)

Males Females

Maximum Maximum entropy entropy Hypothesis d.f. criterion x2 criterion XZ A. One locus 1. General 7 -883 42.96 -640 58.69 2. Additive genes 8 -654 274.22 -492 208.54 B. Polygenic 1. General 6 -533 391.45 -275 42 1.09 2. Additive genes 7 -493 433.15 -244 454.49 3. Infinite general 7 -300 625.94 -49 649.19 4. Infinite additive genes 8 -291 637.33 -44 656.12 C. Mixed major locus/polygenic 1. General 2 -916 0.06* -688 0.42* 2. Additive niajor locus genes 3 -862 55.68 -634 56.68 3. Symmetry A 3 -910 7.77* -690 0.45* 4. Symmetry B 3 -918 0.07* -683 7.12* 5. Symmetry A, additive major 4 -728 191.80 -539 153.73 locus genes 6. Symmetry B, additive major 4 -728 191.80 -539 153.73 locus genes 7. Completely additive 6 -725 199.42 -542 153.97 D. (i) Two unlinked loci 1. General 3 -909 8.69 -687 3.88* 2. Additive loci 5 -909 13.49 -686 8.41 3. Symmetry A 4 -891 28.78 -628 64.46 4. Symmetry B 4 -874 45.81 -657 35.19 5. Zero average dominance 6 -804 120.23 -610 86.42 6. Equal dominance ratio, cou- 6 -795 129.28 -500 196.84 pling 7. Equal dominance ratio, repul- 6 -328 596.43 -76 620.08 sion 8. Equal and additive loci 7 -781 144.78 -503 195.90 9. Completely additive 8 -706 222.13 -454 246.78 D. (ii) Two linked loci 10. General 2 -908 8.23 -685 3.79* 11. Additive loci 4 -907 12.82 -685 7.89 12. Symmetry A 3 -904 14.13 -657 33.71 13. Symmetry B 3 -890 27.63 -663 27.83 14. Zero average dominance 5 -854 67.60 -632 62.77 15. Equal dominance ratio, cou- 5 -884 37.85 -542 152.56 pling 16. Equal dominance ratio, repul- 5 -426 495.73 -173 521.26 sion 17. Equal and additive loci 6 -884 39.76 -643 53.70 18. Completely additive 7 -720 205.56 -522 176.01 .I * P > 0.05. 642 J. M. STOLK ET AL.

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P" Activity (unitwadrenal ghd pair) FIGURE2.-Empirical cumulative probability plots of adrenal gland PNMT activity in male F344 backcross (A) and in male Buf backcross populations (B). The rank of each individual subject's enzyme value, divided by the number of rats in the class plus 1, is plotted as cumulative probability (ordinate) against the observed PNMT activity (solid symbols) together with the theoretical distribution (solid line) based upon the mixed major locus/polygenic hypothesis with symmetry B (Table 4). Measured values for the two male backcross populations are summarized in Table 3. with those in Figures 4 and 5, respectively, it is seen that the means of the middle components are approximately the same, whereas those of the extreme components (especially the top components) are displaced toward the middle components in the F,; this is what is to be expected if there is a major locus segregating in the presence of polygenic modifiers.

DISCUSSION The present study employs statistical methods recently proposed by ELSTON (1 984), which modify and extend the maximum likelihood procedures reported by ELSTON and STEWART(1973), to analyze quantitative data on adrenal gland PNMT activity in F344 and Buf inbred rat strains, their F1, backcross and their RAT ADRENAL PNMT INHERITANCE 643

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PNMT Activity (unitwadrenal gland pair) FIGURE3.-Empirical cumulative probability plots of adrenal gland PNMT activity in female F344 backcross (A) and in female Buf backcross populations (B). The rank of each individual subject’s enzyme value, divided by the number of rats in the class plus 1, is plotted as cumulative probability (ordinate) against the observed PNMT activity (solid symbols) together with the theoretical distribution (solid line) based upon the mixed major locus/polygenic hypothesis with symmetry A (Table 4). Measured values for the two female backcross populations are summarized in Table 3.

FP generations. These statistical methods determined sex-specific transformations under which homogeneity among the cage and random components of variance could not be precluded in the parental and F1 classes (see RESULTS) and support a common basic genetic mechanism underlying adrenal PNMT activity in both sexes in addition to sex-specific genotype-environment interactions. Analyses of the transformed PNMT activity values revealed that only mixed major locus/ polygenic hypotheses could account for the data in the male backcross populations (Table 4) and that the data best fit (as judged by the maximum entropy criterion.being minimal) the mixed major locus/polygenic hypothesis (Figures 4 and 8). Analyses of the transformed PNMT values in females were, at first glance, somewhat less conclusive, since either the general mixed or the general two- locus hypotheses could account for the data (Table 4). It is clear from Figures 5 644 J. M. STOLK ET AL.

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P” Activity (units”a1 gland pair) FIGURE4.-Empirical cumulative probability plots of adrenal gland PNMT activity in male parental and F1 populations. The rank of each individual’s enzyme activity, divided by the number of rats in its class, is plotted as cumulative probability (ordinate) against the observed PNMT activity (solid symbols) together with the theoretical distribution (solid line) based upon the mixed major locus/polygenic hypothesis with symmetry B. Measured values for Buf are plotted in the bottom quarter, for FI in the middle half and for F344 in the top quarter of the graph; these values are summarized in Table 3. and 9 (as well as from an inspection of the estimates obtained in the analysis: the recombination fraction was greater than 0.5 and the recombinant means often were estimated to be very close to the nonrecombinant means) that the fit of the two-locus model to the female data was spurious, reflecting an attempt of the hypothesis to model a certain amount of non-normality rather than an indication of the presence of distinct classes of individuals. This is confirmed by the lack of fit of all but the most general two-locus hypotheses, whereas the mixed hypothesis fits with either symmetry condition (Table 4); fit of a particular mode of inheritance without any restriction whatsoever can lead to meaningless results, because parameters such as means of recombinant individuals may coincide with any outlying observations that are present (ELSTONand STEWART1973). Exam- ination of the maximum likelihood estimates of the parameters of the mixed model indicated that either symmetry condition had only a small effect on the estimates. Thus, although the difference in PNMT activity levels between Buf RAT ADRENAL PNMT INHERITANCE 645

I I 1 I I I I

PNMT Activity (units-I glad pair) FIGURE5.-Empirical cumulative probability plots of adrenal gland PNMT activity in female parental and FI populations. The rank of each individual’s enzyme activity, divided by the number of rats in its class, is plotted as cumulative probability (ordinate) against the observed PNMT activity (solid symbols) together with the theoretical distribution (solid line) based upon the mixed major locus/polygenic hypothesis with symmetry A. Measured values for Buf are plotted in the bottom quarter, for FI in the middle half and for F344 in the top quarter of the graph: these values are summarized in Table 3. and F344 rats cannot be totally accounted for on the basis ofa single locus, there is no doubt that different alleles at a single locus account for much of the difference between the two strains, the remaining difference being accounted for by a small polygenic component. The present results in rats are generally consistent with the conclusions reached by CIARANELLOand AXELROD(1973) for inheritance of mouse adrenal gland PNMT. That is, inheritance of adrenal PNMT in both species is largely accounted for by a single gene locus; coincidentally, the inability of ELSTON (1981) to support the conclusions reached by CIARANELLOet al. (1974) in a statistical reevaluation of their data could be due to either a failure to account for cage and litter effects in the analysis or to the presence of polygenes, in addition to a major locus, accounting for part of the mouse strain differences. Although generally concordant, specific interpretations of adrenal PNMT inheritance in the mouse and rat appear to be quite different. As summarized in 646 J. M. STOLK ET AL. l.0

PNMT Activity (unitwadred gland pair) FIGUREB.-Empirical cumulative probability plot of adrenal gland PNMT activity in male F2rats. The rank of each subject's enzyme value, divided by the number of male rats in the F2 population plus 1, is plotted as cumulative probability (ordinate) against the observed PNMT activity (solid symbols). The solid line indicates the theoretical distribution of enzyme activity predicted from all male subjects, except those in the Fg population, by the mixed major locus/polygenic hypothesis with symmetry B (Table 4). Measured values for the male FP population are summarized in Table 3. the introduction, CIARANELLOet al. (1 974) attributed inherited differences in the BALB/cJ and BALB/cN mouse sublines to a regulatory gene mutation affecting intracellular enzyme degradation; this conclusion later was generalized to regulation of rat adrenal PNMT activity, although not specifically to inheritance of enzyme activity in that species (CIARANELLO,WONC and BERENBEIM 1978). Although the present study did not assess potential differences in adrenal PNMT degradation, the data obtained in the F344 X Buf pedigree most likely are accounted for by inheritance at a structural gene locus. The prominent difference in thermal inactivation between PNMT activity in Buf and F344 adrenal homogenates (Figure 1) strongly supports the existence of structural variants (PAIGEN1971); the small but persistent differences in affinity for the cosubstrate, SAMe (Table 2), may reflect effects of a structural gene locus on biological activity of the enzyme. Although data obtained in the present study suggest that inherited differences in rat adrenal PNMT activity are attributable to structural gene variants, the concept advanced by CIARANELLOet al. (1978), RAT ADRENAL PNMT INHERITANCE 647

-9

-6

.1 -

.3 -

MMl Activity (unitsndrenal gland pair) FIGURE'l.-Empirical cumulative probability plot of adrenal gland PNMT activity in female Fz rats. The rank of each subject's enzyme value, divided by the number of female rats in the Fz population plus 1, is plotted as cumulative probability (ordinate) against the observed PNMT activity (solid symbols). The solid line indicates the theoretical distribution of enzyme activity predicted from all female subjects, except those in the Fz population, by the mixed major locus/polygenic hypothesis with symmetry A (Table 4). Measured values for the female FZ population are summarized in Table 3. that regulation of PNMT proteolysis may be influenced by SAMe, remains viable; that is, degradation rates of the structurally dissimilar PNMT molecules may be different, and SAMe may be important in regulating adrenal PNMT proteolysis in the rat. Preliminary data obtained in our laboratory, for instance, indicate that SAMe protects adrenal PNMT from both inbred strains against thermal inactivation but does not alter the basic between-strains differences in thermolability (GUCHHAITet al. 1983). Detailed studies on PNMT structure in the two parental rat strains are in progress and can be expected to shed further light on the inheritance of adrenal PNMT in the F344 X Buf pedigree.

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Corresponding editor: B. S. WEIR