A C/EBP-binding site in the transferrin promoter is essential for expression in the liver but not the brain of transgenic mice.

The gene for the iron-binding protein transferrin is transcribed at a high level in liver hepatocytes but is also active in several other cell types, including oligodendrocytes in the brain. Enhancer elements between bp -560 and -44 of the transferrin gene promoter specifically activated transcription from a heterologous promoter in transgenic mouse liver and brain. Within this region, a potent cis-acting element between bp -98 and -83 was found to be essential for gene activity in both cultured hepatocytes and transgenic mouse liver. The -98 to -83 element contains a CCAAT sequence and is specifically bound by a nuclear factor from mouse liver that is homologous to rat liver C/EBP (CAAT enhancer-binding protein). Point mutations within this binding site inhibit factor binding and abolish transcription in transfected hepatoma cells. When placed in the context of the 3,000-bp transferrin promoter, the C/EBP binding site mutation causes a complete loss of transcription in transgenic mouse liver; however, transgene expression in the brain of the same animals was unaffected. These results suggest a modular structure for the transferrin promoter and demonstrate that deletions or specific point mutations can be used to generate transgene promoters with an activity more restricted than that of their endogenous counterparts.

Alteration of neurotransmitter phenotype in noradrenergic neurons of transgenic mice.

The normal complement of neurotransmitters in noradrenergic neurons was altered by expressing the structural gene for the enzyme phenylethanolamine-N-methyltransferase (PNMT) under the control of the dopamine-beta-hydroxylase gene promoter in transgenic mice. This resulted in accumulation of large amounts of epinephrine in neurons of the sympathetic nervous system (SNS) and central nervous system (CNS) but did not reduce norepinephrine levels. Adrenalectomy reduced PNMT levels in the SNS and CNS, suggesting that the transgene is positively regulated by adrenal steroids. Epinephrine levels were unaffected by this treatment in the CNS, suggesting that PNMT is not rate limiting for epinephrine synthesis. However, catecholamines were elevated in a sympathetic ganglion and a target tissue of the SNS, perhaps due to up-regulation of tyrosine hydroxylase in response to adrenalectomy. These transgenic mice also reveal a marked difference in the ability of chromaffin cells and neurons to synthesize epinephrine.

A unique mRNA species for a regulatory subunit of cAMP-dependent protein kinase is specifically induced in haploid germ cells.

Cyclic AMP (cAMP) and its action by way of cAMP-dependent protein kinase is important for sperm motility. Previous studies on germ cells have demonstrated a selective decrease in the amount of type I cAMP-dependent protein kinase during spermatid development, and that type II was the major form present in elongating spermatids and in mature sperm. This would indicate activation of a gene in haploid germ cells, encoding a regulatory subunit of type II protein kinase. However, haploid expression of such a gene has so far not been shown. In the present study we demonstrate high-levelled expression of a unique mRNA species for a specific regulatory subunit of type II cAMP-dependent protein kinase at late stages of spermatogenesis, i.e. during spermatid elongation.

Catalepsy induced by combinations of ketamine and morphine: potentiation, antagonism, tolerance and cross-tolerance in the rat.

Previous studies demonstrated that both ketamine and morphine induced analgesia and catalepsy in the rat. Pre-treatment with ketamine produced cross-tolerance to morphine, whereas pretreatment with morphine did not induce cross-tolerance to ketamine but rather augmented the cataleptic response; this augmentation was attributed to residual morphine in the brain. The present studies explored the duration of the loss of righting reflex induced by sub-effective doses of ketamine and morphine, administered simultaneously. There was mutual potentiation between sub-effective doses of ketamine and morphine, but sub-effective doses of ketamine partly antagonized fully-effective doses of morphine. Latency to the loss of righting reflex, rigidity and behavior on recovery, reflected the relative predominance of ketamine or morphine in each combination. Naloxone inhibited the induced cataleptic effects. The degree and time course of development of tolerance to daily administration of sub-effective dose combinations of ketamine and morphine were similar. Rats, tolerant to ketamine-dominant combinations, were cross-tolerant to both drugs, while those tolerant to morphine-dominant combinations were cross-tolerant to morphine but showed either no cross-tolerance or an augmented response to ketamine. While the mutual potentiation, antagonism and tolerance suggest common mechanisms for the induced catalepsy, differences in latency, rigidity and behavior, asymmetry of cross-tolerance and a widely-different ID50 for naloxone would argue against an action at a single opioid site.

Effects of repeated restraint in Japanese quail genetically selected for contrasting adrenocortical responses.

Behavioral and adrenocortical responses to repeated mechanical restraint were compared in 28-day-old to 31-day-old male Japanese quail from two genetic lines divergently selected for reduced (low stress, LS) or exaggerated (high stress, HS) plasma corticosterone (C) responses to brief immobilization. Restraint in a metal crush cage for 5 min elicited immobility and silence in all the birds. Circulating C levels were considerably higher in quail of both lines following restraint than in the undisturbed controls of either line. As expected, both the behavioral and physiological effects were more pronounced in HS than in LS birds. Struggling increased with repeated restraint in HS and LS quail, thus suggesting behavioral habituation to the stressor in both lines. On the other hand, a line effect on the pattern of adrenocortical responses was revealed upon subtracting the change in plasma C concentrations from Day 1 to Day 4 in the undisturbed controls from the corresponding change in restrained birds. Thus, unlike LS quail, in which there were no detectable effects of repeated restraint, the adrenocortical responses of HS birds showed evidence of experience-dependent sensitization. Our results demonstrate the importance of the background genome in determining the patterns of the behavioral and adrenocortical responses elicited by repeated exposure to stressful stimulation. The present results and those of previous studies could be explained in one or both of two ways: that underlying fearfulness is lower in LS than HS quail or that they adopt active or passive coping strategies, respectively. Our findings may also have important implications for poultry welfare and productivity. @ 2000 Elsevier Science Inc.

Timidity in Japanese quail: effects of vitamin C and divergent selection for adrenocortical response.

Male Japanese quail chicks of two genetic lines selected for low (LS) or high (HS) adrenocortical responses to mechanical restraint were housed in mixed-line groups of 24 in four compartments of a multitier brooder battery at 20 days of age. Quail in two of the four compartments were given vitamin C (ascorbyl-2-polyphosphate, APP, 1 g L-ascorbic acid/L) solution for 48 h, whereas the other birds received untreated tap water as usual before they were tested at 23 days of age. At test, each quail was placed individually in a dark, sheltered compartment of an emergence box and allowed 1 min to acclimatise before a door was raised allowing access to an illuminated and exposed area. Vocalisation and the latencies to head and full emergence were then recorded to measure its fear levels. More LS quail vocalised than did HS ones. They also emerged more rapidly from the sheltered compartment into the illuminated one than HS birds. These findings further support our hypothesis that decreased fearfulness has accompanied genetic selection for reduced adrenocortical responsiveness. Treatment with APP reduced the latency to emerge fully into the exposed compartment, and there were no line x treatment interactions. These results suggest that vitamin C supplementation alleviated fearfulness, regardless of existing line differences in this behavioural trait.

Developmental instability in japanese quail genetically selected for contrasting adrenocortical responsiveness.

Differences in developmental instability were assessed with Japanese quail of two lines that had been genetically selected over several generations for reduced (low stress, LS) or exaggerated (high stress, HS) plasma corticosterone response to brief mechanical restraint. At 32 wk of age, three bilateral traits were selected for study in each quail line. The characteristics chosen were length of the metatarsus (shank length, SHL), diameter of the shank (SHD) perpendicular to the spur, and distance between the auditory canal and the nares (face length, FL). Significantly greater bilateral trait size variances were associated with the measurement of SHL (P < 0.0088) and FL (P < 0.0016) in the HS line than in the LS line. SHD variances did not differ (P = 0.22) in quail of the HS and LS lines. These findings suggest that developmental instability (i.e., fluctuating asymmetry, FA) is more pronounced in HS quail than in LS quail. Previous studies have shown that not only do quail of the HS line show greater adrenocortical responsiveness to a wide range of stressors but that they are also more easily frightened than LS birds. Therefore, the line differences in FA found here may reflect the birds' differential responsiveness to chronic social and physical environmental stressors. The present findings also support previous suggestions that measuring asymmetries in bilateral traits could be an additional and valid method of assessing stress and of comparing phenotypic stability in selected populations.

Active immunization of broiler breeder hens with a recombinant chicken inhibin fusion protein enhances egg lay.

We have demonstrated that inoculation of female Coturnix with an inhibin-based immunogen (MBP-cINA521) accelerated puberty and enhanced hen-day egg production (HDEP). Herein, MBP-cINA521, a fusion protein, which consists of the bacterial maltose binding protein (MBP) and a fragment of the infinity-subunit of chicken inhibin (cINA521), was tested for its ability to enhance production performance in broiler breeders. Pullets (Arbor Acres Classic Females; n = 60 birds/treatment group) were given (subcutaneously) 0, 1, 3, or 5 mg of MBP-cINA521 in Freund's complete adjuvant at 20 wk and 4 d of age. Booster immunizations (one-half of the primary dosages) were given at 23 wk of age. The vehicle for controls (CON; no booster) and MBP-cINA521-boosted birds was Freund's incomplete adjuvant. Blood samples were obtained at the end of the trial to assess immunological response to the antigen with a titer ELISA. The onset of puberty was assessed by calculation of the average ages at first lay (FIRST) and at 50% egg production (FIFTY). Cumulative percentage HDEP was determined weekly throughout the laying period (40 wk). Egg weight (EWT) and specific gravity (SG) assessments were made periodically during the trial. Body weight gain (BWG) and mortality (MORT) data were also collected. Significant injection treatment differences (P < 0.01) in inhibin antibody titers were detected according to the following order: low dose = intermediate dose > high dose > CON. A dose of MBP-cINA521 capable of accelerating puberty and increasing overall egg lay was identified. FIRST and FIFTY responses were decreased (P < 0.05) in birds given the intermediate dose (3.0 mg) of MBP-cINA521 when compared to the CON. FIFTY responses were also lower (P < 0.05) than CON responses in those birds given the highest dose (5.0 mg) of the inhibin antigen. Cumulative HDEP was also higher (P < 0.05), beginning at 3 wk of lay and weekly thereafter (P < 0.05, for the remaining 40 wk), in birds given the intermediate immunogen dosage when compared to the CON. By Week 40, an average increase of 9.5% HDEP was realized in birds given 3.0 mg of MBP-cINA521. MORT rates were similar in the CON and in the two lowest MBP-cINA521 treatment groups but were higher (P < 0.05) in those birds given 5.0 mg of the antigen. EWT, SG, and BWG measurements were unaffected by treatment with the inhibin vaccine. In agreement with our findings in quail, immunoneutralization of inhibin enhanced production performance in breeder hens.

Genetic characterization of stress responsiveness in Japanese quail. 1. Analyses of line effects and combining abilities by diallel crosses.

The inheritance of stress responsiveness (line effects and combining abilities) was phenotypically studied in progeny from diallel crosses of randombred (RB) quail and quail selected for exaggerated (high stress, HS) or reduced (low stress, LS) plasma corticosterone (CS) response to brief immobilization. The three genotypes were crossed in a 3 x 3 factorial arrangement of treatments that allowed all possible crosses between RB, LS, and HS males with RB, LS, and HS females. The nine crosses produced 479 progeny. At 28 d of age, quail of each cross were stressed by immobilization, and blood was sampled. Plasma CS was used to estimate stress responsiveness in the progeny of each cross. Estimates of the following genetic effects were made: average post-immobilization plasma CS response within each cross, general combining ability (GCA), and specific combining ability (SCA). Differences (P < 0.05) in plasma CS response to immobilization between crosses involving the LS and HS lines (HS crosses > LS crosses) were commonly observed. There were no significant differences in the means of plasma CS responses between males and females within each of the crosses except for the marginal (P = 0.07) sex difference (male > female) found within the LL cross (LS male with LS female). The GCA estimates were -1.23, -2.89, and 5.08 for the RB, LS, and HS quail, respectively. The SCA was not significant for any diallel mating except HH (P < 0.007). Line effects on plasma CS response were different from zero for the LS and HS lines.

Genetic characterization of stress responsiveness in Japanese quail. 2. Analyses of maternal effects, additive sex linkage effects, heterosis, and heritability by diallel crosses.

Diallel crosses were used to investigate the genetic inheritance of stress responsiveness through examination of population effects in progeny of randombred (RB) quail and quail selected for reduced (low stress, LS) or exaggerated (high stress, HS) plasma corticosterone (CS) response to brief immobilization. The three genotypes were crossed in a 3 x 3 factorial arrangement of treatments that allowed all possible crosses between RB, LS, and HS males with RB, LS, and HS females. The nine crosses produced 479 progeny that were, at 28 d of age, stressed by immobilization, and a sample of blood was collected. The following quantitative genetic parameters were estimated for plasma CS: heritability, heterosis, maternal effects, sex-linkage effects, and heterosis due to the sex chromosomes. Genotypic-phenotypic correlations within RB quail and quail of the two selected lines were also obtained. Maternal effects were different (P < 0.05) from zero for the plasma CS responses of all three genotypes. Additive sex linkage effects on plasma CS responses were variable and of much less importance than maternal effects. The correlations between plasma CS response and genotype were: 0.22 for RB quail and 0.37 and 0.55 for quail of the LS and HS lines, respectively. Heterosis effects were low and most likely due to either an increase in homozygozity of genes on the sex chromosomes or an increase of maternal effects within reciprocal crosses. Heritability, estimated for the post-immobilization plasma CS response, was 0.05 for RB quail and 0.14 and 0.30 for quail of the LS and HS lines, respectively.

Factors affecting ostrich egg hatchability.

Ostrich eggs often have low hatchability (HATCH) rates because they do not lose sufficient weight during incubation. Because egg size, eggshell porosity and thickness (THICK), and length of preincubation egg storage are known to affect egg weight loss during incubation (EWL) and HATCH of chicken eggs, these factors were examined using ostrich eggs. The effects of eggshell porosity (number of large pores per cm2 of shell; LP); and THICK on EWL and HATCH were assessed by categorizing the eggs as having either low, intermediate, or high LP or low, intermediate, or high THICK. Mean EWL was higher (P<0.05) in eggs of the high LP group when compared with eggs in either the low or intermediate LP groups that lost similar amounts of weight during incubation. Mean HATCH was also higher (more than 25%; P<0.10) in eggs with high LP when compared with the HATCH found in eggs having low LP. Eggs from the intermediate LP group had an intermediate HATCH response. Moreover, numbers of LP were positively correlated to both EWL (r2 = 0.64; P<0.0001) and HATCH (r2 = 0.25; P<0.03). Inverse relationships existed between THICK and EWL and between THICK and HATCH according to the order (P< 0.05): eggs of low THICK, highest mean EWL and HATCH > eggs of intermediate THICK, intermediate mean EWL and HATCH > eggs of highest THICK, lowest mean EWL and HATCH. Shell thickness was not correlated to either EWL or HATCH. The influence of egg size on mean LP, THICK, EWL, HATCH, and chick weight (CWT) was assessed. Although THICK was unaffected by egg size, higher LP (P<0.10), EWL (P<0.05), and HATCH (P<0.10) were found in medium-sized eggs when compared with either small or large eggs. The CWT was associated with egg size (P<0.05) according to the order: large eggs, highest CWT > medium eggs, intermediate CWT > small eggs, lowest CWT. Neither EWL nor HATCH was affected by length of preincubation egg storage. Collectively, our findings suggest that 1) ostrich eggs that possess low LP and increased THICK hatched poorly, 2) intermediate-sized eggs hatch best, 3) large eggs produced large chicks, and 4) ostrich eggs can be stored under conditions typically used in the poultry industry for a minimum of 10 d without negatively impacting HATCH.

T-maze behavior and early egg production in Japanese quail selected for contrasting adrenocortical responsiveness.

Broiler chicks that traverse a T-maze quickly to reinstate contact with their companions (HP, high performance) are known to grow faster, be more social, and exhibit a reduced plasma corticosterone (B) response to acute stress than slower (LP, low performance) chicks. Genetic lines of Japanese quail selected for reduced (LS, low stress) or exaggerated (HS, high stress) plasma B response to brief restraint also differ in sociality and performance. In the present study, we asked if divergence in early T-maze behavior was associated with differential attainment of puberty and early egg production in these lines. At 3 d of age, LS and HS quail were categorized as HP or LP birds based on running times in a T-maze. Thus, there were four treatment combinations: HP-LS, HP-HS, LP-LS, and LP-HS birds. Daily egg records were kept for 8 wk. The average ages at first egg lay (FIRST), at 25% egg production (A25% EP), and weekly and cumulative hen-day egg production (HDEP) where calculated. Daily egg weight (EWT) data were also collected, and BW measures were made at the end of the trial. Mean FIRST and A25% EP responses were lower (P < 0.02) and cumulative HDEP was greater (P < 0.04) in HP than in LP quail. Despite considerable numerical reductions in FIRST and A25% EP, as well as 5% elevation in cumulative HDEP in LS quail, line differences in these variables were not significant. On the other hand, FIRST and A25% EP were reduced (P < 0.05) in HP-LS quail when compared to LP-HS ones, whereas HP-HS and LP-LS quail showed intermediate and similar responses that did not differ from the other two treatment groups. Mean cumulative HDEP findings for the interactive effect of performance category with line mimicked these puberty findings. EWT and BW measures were not affected by any of the treatments or their interactions. Our results suggest that rapid negotiation of the T-maze by quail chicks is associated with accelerated puberty and increased HDEP in quail of two genetically diverse lines. This effect is particularly evident in quail selected for reduced adrenocortical responsiveness, suggesting possible additive effects.

Struggling behavior in shackled male and female broiler chickens.

Anecdotal evidence suggests that the struggling behavior of shackled broiler chickens may be positively related to compression of the shank and the probable associated discomfort: birds with large shanks tend to struggle more violently than do those with smaller shanks. Males are generally heavier and have thicker shanks than females. Therefore, we tested the hypothesis that, because the leg gaps of shackles are fixed in size, male broilers would struggle more than females. At 42 d of age, 264 floor-reared broilers were cooped in groups of 12 (six males and six females) and were transported from the university farm to the abattoir. Eighty of these served as test birds (n = 40/sex) and were shackled on a moving processing line with a bird of randomly selected sex on either side. Upon shackling, the latencies to struggle, numbers of struggling bouts, and total time spent struggling were recorded during a 1-min test period. Subsequently, the BW and circumference of the right shank (CRS) of each test bird were measured. Male birds were heavier and had thicker shanks than females (both P < 0.0001); they also struggled sooner (P < 0.01) and longer (P < 0.008). When data from males and females were pooled, CRS was negatively correlated with latency to struggle (r = -0.30; P < 0.006) and positively associated with SB (r = 0.23; P < 0.04) and total time spent struggling (r = 0.23; P < 0.04). However, there were no detectable correlations within sex. Body weight was not significantly correlated with any of the struggling behavior measures. Although other gender-related factors may be influential, an interpretation of our findings based on sex differences in CRS seems the most parsimonious. We conclude that use of shackles of fixed leg-gap size may contribute to increased struggling behavior in male broilers.

Active immunization of Japanese quail hens with a recombinant chicken inhibin fusion protein enhances production performance.

The effects of active immunization against inhibin on production performance in female Japanese quail (Coturnix coturnix japonica) were assessed in two separate trials using an MBP-cINA521 fusion protein as an immunogen. The fusion protein, MBP-cINA521, consisted of the bacterial maltose binding protein (MBP) and a truncated form of the mature alpha-subunit of chicken inhibin (cINA521). MBP-cINA1521 was constructed by: 1) excising a 521-bp PstI fragment from a chicken inhibin alpha-subunit cDNA (cINA6; gift of P. A. Johnson), 2) cloning this fragment, which encodes all but the first 11 amino acid residues of the mature alpha-subunit, into the pMal-c2 vector of the MBP fusion expression system, and 3) expressing the fusion protein (MBP-cINA521) from the Escherichia coli and purifying it using affinity chromatography. In each trial, quail were randomly and equally assigned to one of two injection treatments as follows: 1) MBP-cINA521 in Freund's adjuvant, or 2) Freund's adjuvant (vehicular controls; CON). All immunizations were given subcutaneously and Freund's complete and incomplete adjuvant were used for primary and booster injections, respectively. In Trial 1, birds were given a primary challenge of 0.2 mg MBP-cINA521 per bird at 25 d of age, followed by booster immunizations (0.1 mg MBP-cINA521 per bird) at 33, 40, 47, 54 and 61 d of age and every 35 d thereafter. The CON birds received vehicular immunizations at the same time intervals. In Trial 2, birds treated with MBP-cINA521 received a primary challenge of 0.2 mg MBP-cINA521 per bird at 26 d of age, followed by booster immunizations (0.1 mg MBP-cINA521 per bird) using the same schedule as that used in Trial 1, with the exception that no boosters were given after 61 d of age. The CON birds received vehicular immunizations at the same time intervals. Collection of production performance data was initiated coincident with the laying of the first egg in each trial (i.e., beginning at 41 and 44 d of age for Trials 1 and 2, respectively) and continued for 30 1-wk periods of lay. Combined data from Trials 1 and 2 indicated that the mean +/- SE age at first egg lay was markedly decreased (P < 0.005) in MBP-cINA521-treated quail (53.4 +/- 0.9 d of age) when compared to the CON (57.6 +/- 1.3 d of age). Likewise, the mean +/- SE age at 50% egg production was reduced (P < 0.03) in quail immunized against inhibin (65.4 +/- 2.1 d of age) when compared to the CON (77.6 +/- 4.7 d of age). Total hen-day egg production was also higher (P < 0.05, Trial 1; P < 0.01, Trial 2) in MBP-cINA521-treated quail (88.7 +/- 1.4%, Trial 1; 90.1 +/- 1.2%, Trial 2) than in the CON birds (81.9 +/- 2.9%, Trial 1; 73.6 +/- 6.5%, Trial 2). Collectively, these findings provide evidence that inhibin immunoneutralization accelerated puberty and enhanced hen-day egg production during a 30-wk period of egg lay in Japanese quail.

Genetic characterization of a brain-specific form of the type I regulatory subunit of cAMP-dependent protein kinase.

An isoform (RI beta) of the regulatory type I subunit gene of cAMP-dependent protein kinase (EC 2.7.1.37) has been characterized in mouse. The open reading frame of the RI beta cDNA is 72% identical in nucleotide sequence with the previously cloned RI gene, now referred to as RI alpha. Both genes code for a protein of 380 amino acids and their proteins are 82% identical in amino acid sequence. Sequence similarity is highest in the regions that form the pseudosubstrate-binding site of the catalytic subunit and the two cAMP binding domains. The amino-terminal portion shows the greatest dissimilarity, suggesting that the isoforms may differ in their dimerization properties or interaction with other proteins. In contrast to RI alpha, which is constitutively expressed in all tissues, RI beta is expressed in a highly tissue-specific manner. Brain and spinal cord contained significant levels of RI beta mRNA, testis RNA gave a detectable signal, and all other tissues tested were negative. Expression of a RI beta cDNA in NIH 3T3 cells resulted in the appearance of a RI subunit protein that migrated more slowly than RI alpha after NaDodSO4/PAGE. The native form of RI beta in brain could also be distinguished from RI alpha by its abnormal migration on NaDodSO4/PAGE. RI beta protein produced in 3T3 cells was shown to be functional by its ability to form a cAMP-dependent holoenzyme with the catalytic subunit.

Holoenzymes of cAMP-dependent protein kinase containing the neural form of type I regulatory subunit have an increased sensitivity to cyclic nucleotides.

Specific isoforms of the cAMP-dependent protein kinase are preferentially expressed within discrete neuronal regions in mouse brain (Cadd and McKnight (1989) Neuron 3, 71-79) suggesting that these subunits might have different functional properties. We have used recombinant techniques to express and purify the type I regulatory subunits, RI alpha and RI beta, the catalytic subunits C alpha and C beta, and then reconstituted holoenzymes with the various combinations of R and C subunits. The ability of the subunits to form inactive holoenzymes and then to be activated in the presence of cyclic nucleotides was examined. Holoenzymes containing C beta had essentially the same activation properties exhibited by C alpha holoenzymes. However, the presence of the neural form of RI, RI beta, led to formation of a holoenzyme which was activated at a 3-7-fold lower concentration of cyclic nucleotides compared to holoenzymes containing RI alpha. Expression of the RI beta protein in discrete regions of the central nervous system may provide a mechanism for increasing the sensitivity of the kinase to what would otherwise be subthreshold levels of stimulation. Two mutant forms of RI beta were constructed that converted the RI beta sequence to that of RI alpha at position 98 (RI beta Ala) or positions 98 and 99 (RI beta Ala/Ile). These sequences form part of a pseudosubstrate site thought to interact with the C subunit. Wild type and mutant R subunits were combined in vitro with purified bovine C subunits and half maximal activation constants (Ka) were determined with cyclic nucleotides. Holoenzymes containing RI beta Ala and RI beta Ala/Ile gave Ka values which were higher than wild type RI beta, with the double mutant shifting toward the Ka value of RI alpha holoenzymes by about 30%. These results suggest that amino acid differences in the pseudosubstrate site may account for some, but not all, of the increased sensitivity to cyclic nucleotides exhibited by RI beta.

Isolation and characterization of bovine cardiac muscle cGMP-inhibited phosphodiesterase: a receptor for new cardiotonic drugs.

We have identified and highly purified a "low Km" cAMP phosphodiesterase from bovine cardiac muscle. This phosphodiesterase was inhibited by low concentrations of cGMP and has, therefore, been temporarily designated as cGMP-inhibited phosphodiesterase. After a 16,000-fold increase in specific activity, the highly purified enzyme had a specific activity of 6 mumol/min-mg and contained three major polypeptides. Initial data indicated that all of these polypeptides were derived from a single common precursor by proteolysis. We used this enzyme preparation to generate polyclonal antisera and monoclonal antibodies directed against the "low Km" phosphodiesterase. Immunoadsorption and immunoblot analysis allowed us to identify and isolate several molecular weight species of phosphodiesterase, including a larger form than previously reported for any purified low Km phosphodiesterase. This large form of the enzyme had a subunit molecular weight of approximately 110,000 and was the only one seen in fresh extracts of cardiac muscle. Full catalytic activity was recovered in the phosphodiesterase-antibody complex and enzyme prepared by immunoprecipitation exhibited Michaelis-Menten kinetics for cAMP hydrolysis and for inhibition by cGMP. The Km for cAMP hydrolysis was 0.15 microM and the Ki for cGMP inhibition of cAMP hydrolysis was 0.06 microM. This immunoprecipitation approach also allowed us to determine that the enzyme was phosphorylated on serine residues by cAMP-dependent protein kinase, and that the low Km, cGMP-inhibited phosphodiesterase was selectively inhibited by several new cardiotonic agents. Milrinone, amrinone, and fenoximone were highly selective inhibitors of this isozyme, and the relative affinities of these inhibitors were consistent with their order of potency as positive inotropic agents. These studies suggest that the cGMP-inhibited phosphodiesterase is a receptor for several new cardiotonic drugs.

Inhibition of intracellular cAMP-dependent protein kinase using mutant genes of the regulatory type I subunit.

Expression vectors were constructed that code for mutated forms of the regulatory type 1 subunit (RI) of cyclic AMP-dependent protein kinase. These mutations alter a specific amino acid which is present in each of two homologous cAMP-binding domains of the RI protein. When these expression vectors were introduced into NIH 3T3 and Y1 adrenocortical tumor cells a mutant RI protein was produced that acted in a dominant fashion to cause a 20-400-fold inhibition of cAMP-dependent protein kinase activation. In addition, processes controlled by cAMP in adrenal cells were blocked; cells became resistant to the growth-inhibitory effects of cAMP and defective in steroid synthesis. Expression of mutant RI genes in cells provides a specific means to explore the role of cAMP and protein phosphorylation in the process of intracellular signalling.

Ketamine- and morphine-induced analgesia and catalepsy. I. Tolerance, cross-tolerance, potentiation, residual morphine levels and naloxone action in the rat.

The effects of ketamine and morphine on pain perception and catalepsy were compared in rats. Analgesia, as measured by the latency to withdrawal of the tail from a 55 degrees C water bath (tail-flick latency difference, TFLD), was produced by both ketamine and morphine, but at widely different doses, and in each case the effect was reversed by naloxone. Catalepsy, measured by the duration of loss of righting reflex (DLRR) in catatonic animals, was induced by larger doses of both ketamine and morphine and in each case was reduced by a larger dose of naloxone. DLRR and TFLD tolerance developed rapidly and with a similar time course after daily doses of ketamine or morphine. Rats tolerant to the DLRR effect of ketamine showed cross-tolerance to morphine. Rats tolerant to the DLRR effect of morphine did not show cross-tolerance to ketamine when administered the following day; instead, these rats showed potentiation of the ketamine-induced DLRR. The degree of potentiation noted 24 hr after a single or multiple daily doses of 45 mg/kg of morphine is the same as that seen when 2 mg/kg of morphine is given simultaneously with ketamine. The residual brain level of morphine 24 hr after 45 mg/kg is similar to the level 1 hr after a 2-mg/kg dose. The augmented ketamine response in morphine-tolerant rats is postulated to be a result of residual morphine still present in the brain 24 hr after the last DLRR-inducing dose of morphine.(ABSTRACT TRUNCATED AT 250 WORDS)