Harold G. Klemcke

Plasma adrenocorticotropic hormone and cortisol in pigs: effects of time of day on basal and stressor-altered concentrations.

Abstract An initial study was conducted to establish the presence in plasma of diurnal rhythms of immunoreactive porcine adrenocorticotropic hormone (pACTH) and cortisol in castrated male pigs (barrows). Fourteen barrows with jugular catheters were bled at 6-hr intervals for 24 hr. Significant changes in plasma pACTH were evident with peak levels (61 ± 6 pg/ml) at 0100–0700 hr and a trough (38 ± 4 pg/ml) at 1900 hr. Changes (P < 0.05) in plasma cortisol were also present in barrows with a peak (44 ± 6 ng/ml) at 0700 hr and a trough (21 ± 5 ng/ml) at 1900 hr. Plasma norepinephrine and epinephrine were measured at the same time intervals and did not differ among hours. In these unstressed pigs the ratio cortisol/log10 pACTH at 0700 hr (25.3 ± 3.0) was greater than the ratio at 1900 hr (12.9 ± 2.7). Sequential blood samples were subsequently taken on four of the barrows 12 and 26 days later. Plasma pACTH was variable among pigs and did not differ among hours. Plasma cortisol on both dates was greater (P < 0.05) in the morning (0100 or 0700 hr) than at 1900 hr. The ratio cortisol/log10 pACTH at 0700 hr was repeatedly greater than at 1900 hr. A second study was conducted to determine whether plasma pACTH and cortisol responses to mild (32°C for 2 hr) or strong (20-min restraint) stressors were dependent on the time of day of stressor application (0800 hr, AM; 1600 hr, PM). Response-associated parameters (maximum concentration, maximum incremental concentration, and integrated response) for pACTH and cortisol did not differ between AM and PM. However, a qualitative difference existed between the AM and PM plasma pACTH responses to restraint +32°C wherein the AM response consisted of a single prolonged surge, and the PM response of an initial major peak followed by a second significant minor peak. A suggested explanation is that the initial 20-min restraint stressor potentiated the hypothalamic-hypophyseal response to 32°C. These studies are the first direct measurements which suggest the presence of diurnal changes in plasma ACTH and cortisol in barrows. The studies also indicate for barrows an absence of diurnal changes in plasma epinephrine and norepinephrine. The responsiveness of the pituitary-adrenocortical axis to stressors did not exhibit quantitative diurnal changes at the time periods measured. However, it is hypothesized that the repeatable AM-PM difference in the ratio cortisol/log10 ACTH reflects a diurnal change in adrenal responsiveness to ACTH in unstressed pigs.

Stressor-associated alterations in porcine plasma prolactin

Abstract Experiments were conducted to determine effects of restraint and thermal stressors on plasma prolactin (PRL) in castrated male pigs. A single 20-min restraining period in a restraining cage which prevented both movement and injury increased (P < 0.05) plasma PRL when applied at either 0800 or 1600 hr. Exposure to 32°C at 0800-1000 hr or at 1600-1800 hr produced more moderate increases (P < 0.05). A combination of 20 min restraint and 2 hr at 32°C produced a response similar to restraint alone. Twenty minutes after stressor application plasma PRL concentrations in pigs exposed to restraint or restraint +32°C at 1600 h were greater (P < 0.05) than concentrations measured in all other treatment groups at that time interval. However, there were no statistically significant differences in additional quantitative indices of the plasma PRL responses (maximal level, maximal change, or integrated response above basal levels) among restraint, 32°C, or restraint +32°C, nor between morning and afternoon applications of treatment. Such data do not provide, therefore, any strong evidence for stressor-dependent or circadian differences in plasma PRL response. A second study subjected castrated male pigs to 20°C (controls), 20±12°C (cyclic temperature, sine wave variation), 5°C constant, and 5 ±12°C cyclic for 20 days. After 6 days exposure to 5°C constant or 5 ± 12°C cyclic there were decreases (P < 0.05) of 59 and 67% respectively in plasma PRL when compared either with pretreatment levels or with levels in pigs at 20 or 20± 12°C. There were no differences in PRL responses between cyclic vs constant temperatures. These results are the first to indicate that plasma PRL in pigs is affected by acute restraint and thermal stressors.

Receptor binding and Nb2 cell mitogenic activities of glycosylated vs. unglycosylated porcine prolactin

Purified fractions of glycosylated (pGPrl) and unglycosylated (pUGPrl) porcine prolactin were prepared by affinity chromatography on Concanavalin A-Sepharose. The relative binding activities of these two forms of prolactin for receptors from porcine mammary, adrenal cortex and rabbit mammary, as well as their Nb2 cell mitogenic activity were determined. In both the porcine mammary and adrenal cortex receptor binding assays pGPrl had a 2-3 fold lower activity than pUGPrl. In the rabbit mammary binding assay pGPrl had a about a 5 fold lower activity than pUGPrl. Similarly, pGPrl had only about 20% of the activity of pUGPrl in the Nb2 cell proliferation assay.

Porcine adrenal prolactin receptors: characterization, changes during neonatal development and effects of hypoprolactinemia.

1. Adrenal prolactin (PRL) receptors were identified within the adrenal cortex of pigs (Sus domesticus), and found to be located specifically on isolated zona fasciculata/reticularis cells (6437 sites per cell). 2. These PRL receptors were associated with binding to [125I]-oPRL which was characterized as being time and temperature dependent, specific for PRL, saturable, of high affinity (Ka = 10(10)/M) with a single class of binding sites, and irreversible except under extreme conditions. 3. The concentrations (fmol/mg protein) of PRL receptors decreased by 35% (P less than 0.05) between 3 and 10 days of age, and subsequently remained constant until 30 days of age. Total content (fmol/paired adrenals) increased progressively (2-fold, P less than 0.05) between 3 and 30 days of age. 4. Short-term (less than 16 hr) and prolonged (7 weeks) hypoprolactinemia (46-64% of control levels, P less than 0.05) were not associated with changes in numbers of porcine adrenal unoccupied PRL receptors.

Ontogeny of glycosylated and nonglycosylated forms of prolactin and growth hormone in porcine pituitary during fetal life.

Abstract Although prolactin (PRL) and growth hormone (GH) were long considered to be nonglycoprotein hormones of the pituitary, glycosylated forms of these hormones have nevertheless been discovered in recent years. We determined the ontogeny of glycosylated and nonglycosylated PRL and GH during the fetal life of the pig, an animal in whose pituitary the glycosylated variant of PRL has been found in high (40%) concentrations. Swine fetuses of both sexes from lean and obese animals of Duroc x Yorkshire crosses were examined at 60, 75, 90, and 105 days of age. No appreciable differences related to sex or phenotype were noted in any of the parameters measured; therefore, data for all animals within an age group were combined. Such averages revealed considerable amounts of GH in the fetal pituitary as early as 60 days of age, whereas PRL, although detectable by radioimmunoassay and immunoblotting at all ages tested, was not present in significant amounts until 105 days of age. From its first appearance, however, almost 70% of the PRL synthesized in the fetal pituitary was of the glycosylated type. In contrast to PRL, both the glycosylated and nonglycosylated forms of GH showed a steady rate of increase throughout the observation period. The immunoblotting analyses also revealed in the fetal pituitary several intensely staining 8-to 12-kDa PRL-immunoreactive peptides of unknown identity. The occurrence of significantly greater concentrations of glycosylated PRL than of non-glycosylated PRL in the fetal pituitary during late gestation offers new possibilities for the role of this hormone in the development of the swine fetus.

Functioning of the porcine pituitary-adrenocortical axis during neonatal development

A study was conducted with neonatal boars to measure age-related changes in functioning of the pituitary-adrenocortical axis. Pigs were randomly assigned to control (n = 7-10/age) or treated (1-min restraint, n = 9-11/age) groups to be sampled at either 12, 19, or 26 days of age. Blood samples were taken via catheter 10 min before and 3, 10, and 20 min after restraint or at similar time intervals in controls. One day later, pigs were killed and adrenal glands obtained for ACTH receptor measurements. Basal plasma ACTH concentrations were greatest (p = 0.035) on day 12 when compared with later ages, but basal plasma cortisol concentrations were comparable at the three ages. Compared with controls, restraint elevated incremental plasma ACTH and cortisol responses at each age (p < 0.004). On day 12, maximal plasma ACTH (p = 0.0006) and incremental cortisol (p < 0.006) responses to restraint were greater than at later ages. Binding to adrenal ACTH receptors was greatest (p < 0.05) at day 13, which may help explain the apparently increased in vivo response of the adrenal gland to ACTH at this time. Restrained pigs had increased growth rates with increasing age (p = 0.016) whereas growth rates for control pigs did not differ with age. At day 27, 24 h after the 1-min restraint, body weights of restrained pigs exceeded those of control pigs (p = 0.045). At day 20, adrenal DNA and protein in pigs restrained 24 h previously were greater than in control pigs (p < 0.05). These data suggest age-related changes in functioning of the pituitary-adrenal axis in neonatal boars, and an absence of period during neonatal life when the porcine pituitary adrenocortical axis cannot respond to a stressor. The data also indicate both rapid and long-term responses of the adrenal to a very modest stressor and suggest an extreme sensitivity of neonatal pigs to environmental perturbations.

Hormonal and stressor-associated changes in porcine adrenocortical cholesterol ester hydrolase activity

Cholesterol ester hydrolase (CEH) activity was characterized in the porcine adrenal gland and experiments conducted to determine the nature of its hormonal regulation. CEH activity was studied in the 14,000 gmax pellet (F4) and in the 192,000 gmax supernatant (F6). Characteristics associated with pH optima, product formation with time, linearity with increasing protein concentration, and equilibration of exogenous cholesterol esters added in acetone with endogenous cholesterol esters were determined. Scatchard analyses of saturation data demonstrated two-site models, which indicated the presence of lower velocity lower Km enzymes (catalytic sites) (L-VKm) and higher velocity higher Km enzymes (catalytic sites) (H-VKm) in both subcellular fractions. Neither ACTH (0.4 micrograms/kg body weight) nor 30-min restraint affected CEH activities at 0.5, 2, and 5 h after injection or initiation of restraint. However, 1 h after a longer restraint period (45 min), F4 H-VKm CEH activity increased concomitantly with decreased F6 L-VKm (P = 0.003). More modest increases in F4 H-VKm (P = 0.03) were still apparent 1 h after the last of nine daily 45-min restraints. Bromocriptine (CB154, a dopamine agonist) administration for 6 days (9.6 mg/daily) reduced plasma prolactin (PRL) by 53% (P < 0.05), but had no effect on CEH activities. ACTH treatment to CB154-induced hypoprolactinemic barrows dramatically reduced F4 (63%) and F6 (49%) L-VKm CEH activity (P = 0.03). These data are the first concerned with regulation of adrenal CEH activity in swine, and are the first to evaluate in vivo treatments on in vitro CEH activity in any species evolutionarily higher than rodents. In vivo regulation of porcine adrenal CEH activity appears complex. Stressor-associated hormonal perturbations apparently must surpass a certain threshold of duration and/or magnitude before they alter CEH activity. Differing Km and Vmax of CEH within and between the two subcellular fractions studied and the differential responses to restraint stressor suggest that as many as four different enzymes with CEH activity are involved. Additionally, the combined effect of ACTH and CB154-induced hypoprolactinemia argues for an interrelated modulatory function of ACTH and PRL (or dopamine) on specific porcine adrenal CEH activities.

Ontogenetic changes in porcine adrenocortical adrenocorticotropic hormone receptors

1. Binding of [125I]ACTH(1-38) analog to adrenal receptors was measured in fetal pigs (Sus domesticus) at 15-day intervals from midpregnancy (60 days) to near term (105 days; pregnancy length 114 days). 2. Binding was greatest at day 60 (0.42 +/- 0.03 fmol/200 micrograms protein or 0.50 +/- 0.08 fmol/50 micrograms DNA), and least at day 105 (0.13 +/- 0.03 fmol/200 micrograms protein or 0.16 +/- 0.04 fmol/50 micrograms DNA). Total adrenal binding was constant (0.61 +/- 0.02 fmol/paired adrenals). 3. Scatchard analyses at day 60 and day 105 showed comparable apparent affinities of ACTH receptors (Ka day 60 = 1.51 +/- 0.72 x 10(9) M-1 vs Ka day 105 = 1.94 +/- 0.78 x 10(9) M-1). 4. DNA per paired adrenals and membrane-associated protein increased 1.6-fold, providing a constant protein: DNA ratio. Concentrations of adrenal cortisol were constant from 60 to 90 days of gestation age but increased dramatically by day 105. 5. These data suggest that during 60-105 days of gestation age the number of ACTH receptors per cell is reduced.

Influence of plasma prolactin concentrations and age on neonatal immune function

Prolactin (PRL) plays an immunomodulatory role in adult animals. However, little information is available concerning PRLs effects on neonatal immune function. Experiments were designed to evaluate the effects of hyper- and hypoprolactinemia and age on immunity in neonatal pigs. In Experiment 1, 4-day-old pigs were treated with bromocriptine, an inhibitor of PRL release or vehicle for 1, 2, or 3 weeks. Mitogen-induced lymphocyte proliferation was evaluated in splenic lymphocytes at 11, 18, and 25 days of age. In Experiment 2, PRL or vehicle were administered to 6-day-old pigs for 2 weeks and lymphocyte proliferation, in vitro interleukin 2 (IL-2) and plaque forming cell (PFC) assays were performed. Bromocriptine administration suppressed (P = .0001) plasma PRL concentrations and enhanced concanavalin A-(ConA) and pokeweed mitogen-(PWM) stimulated proliferation of lymphocytes (P = .09). In contrast, PRL administration increased circulating plasma PRL concentrations (P < .03), decreased lymphocyte proliferation (P = .08) and suppressed the numbers of PFC/mg DNA (3.5 ± 1.6) vs control pigs (8.7 ± 3.3). Both in vitro IL-2 production and PWM-induced proliferation was decreased (P = .0004 and P = .002, respectively) in older vs younger pigs. The results of these studies suggest that PRL may limit specific aspects of immune system function in neonatal pigs.