Pig Thyroid Research Articles

Iodothyronine content in the pig thyroid gland

1. 1. An analysis has been carried out on the contents and reciprocal proportions of three principal iodothyronines (T 4, T 3 and rT 3) in the thyroids of fed and fasted piglets of 8–10 wk and in adult pigs. 2. 2. The mean T 4 concentration averaged 62.0 ± nmol/100 mg wet tissue (in adults: 18.5 ± 4.3 nmol/100 mg tissues); T 3, 9.5 ± 0.9 nmol/100 mg tissue (in adults: 1.58 ± 0.2 nmol/100 mg tissue); rT 3, 3.0 ± 0.3 nmol/100 mg tissue. 3. 3. The reciprocal ratios of the hormones in the piglets' thyroids were: for T 3: T 4, 0.150 (in adults, 0.114) and for rT 3: T 4, 0.050 (in adults, 0.023). 4. 4. Mean T 3: T 3: rT 3 ratio in piglets and adult pigs was 20.5:3.1:1 and 66.1:5.6:1, respectively. 5. 5. The results from all examined iodothyronines, show the higher absolute concentration in piglets' than in adult pigs' thyroid tissue, while the reciprocal proportions of the hormones reveal smaller T 4 thyroid contents (comparing with T 3 and rT 3) in piglets than in adults. 6. 6. No changes of absolute thyroidal contents or reciprocal ratios of the iodothyronines were observed in fed and fasted piglets. 7. 7. In a comparison, the pig thyroid contains more triiodothyronine and a higher ratio T 3: T 4 than that in some other species.

Iodide accumulation into thyroid Golgi vesicles.

Pig thyroid Golgi vesicles incubated in a suitable medium were able to concentrate iodide from the medium. This trapping required the integrity of the vesicles, was time- and temperature-dependent, and was inhibited by a competitive inhibitor of iodide active transport (perchlorate), suggesting a facilitated transport mechanism.

Characterization of monoclonal antibodies raised against solubilized thyrotropin receptors in a cytochemical bioassay for thyroid stimulators.

Selected clones of monoclonal antibodies from mice immunized with solubilized preparations of bovine TSH receptors have been characterized in a cytochemical bioassay (CBA) for thyroid stimulators. This assay is based upon quantification of changes in naphthylamidase activity of sections of guinea pig thyroids with use of a chromogenic substrate. Monoclonal 22A6 is a thyroid-stimulating antibody directed at a site within the TSH receptor. Thus, although it is a weak inhibitor of 125I-TSH binding to thyroid membranes, 22A6 is inhibited from binding to membranes by TSH, exhibits a more than additive agonist effect on adenylate cyclase activity when tested at low TSH concentrations in thyroid cells, and is a competitive antagonist of TSH enhancement of adenylate cyclase activity at high TSH concentrations. In the CBA, 22A6 is a stimulator whose maximal activity is obtained with 77 pg/ml (3-min exposure). Dose-response curves of a long acting thyroid stimulator (LATS)-B standard and 22A6 have slopes which are not significantly different; as anticipated, the response to LATS-B is inhibited by antihuman immunoglobulin G (IgG) and that due to 22A6 by antimouse IgG. In contrast to 22A6, monoclonal 11E8 is a relatively potent inhibitor of 125I-TSH binding as well as TSH stimulation of adenylate cyclase activity, while failing to act as a stimulator itself. 11E8 is itself inactive as a stimulator in the CBA over a wide dose range; it does, however, inhibit TSH stimulation in the CBA. This inhibition is abolished by antimouse IgG. The transient peak of response observed in time courses to TSH occurs later in the presence of 11E8. Unlike its effect on TSH 11E8 shows relatively low potency (greater than 10,000-fold lower) when inhibiting stimulation by the thyroid stimulating antibodies, 22A6 or LATS-B. Since this difference cannot be explained by quantitative differences in the ability of 22A6 or 11E8 to bind to thyroid membranes, the CBA data suggest that the stimulating antibodies, 22A6 and LATS-B, may interact with different determinants on TSH receptors then either TSH or the blocking antibody, 11E8. This also implies that in Graves' disease blocking antibodies may be incompletely expressed in the presence of stimulating antibodies, although they may be potent inhibitors of TSH binding, as measured in receptor assays.

Preparation of isolated pig thyroid follicles with normal polarity and function

Journal Article Preparation of isolated pig thyroid follicles with normal polarity and function Get access W Greil, W Greil Medizinische Klinik InnenStadt, Universität München Search for other works by this author on: Oxford Academic Google Scholar R Gärtner, R Gärtner Medizinische Klinik InnenStadt, Universität München Search for other works by this author on: Oxford Academic Google Scholar D STübner, D STübner Medizinische Klinik InnenStadt, Universität München Search for other works by this author on: Oxford Academic Google Scholar W Permanetter, W Permanetter Medizinische Klinik InnenStadt, Universität MünchenPathologisches Institut, Universität München Search for other works by this author on: Oxford Academic Google Scholar K Horn K Horn Medizinische Klinik InnenStadt, Universität München Search for other works by this author on: Oxford Academic Google Scholar Acta Endocrinologica (Norway), Volume 104, Issue 4_Supplement_b, Dec 1984, Pages S49–S50, https://doi.org/10.1530/acta.0.107S049 Published: 01 December 1984

Generation of H2O2 in isolated porcine thyroid follicles.

Generation of H2O2, an essential component in thyroid hormone synthesis, was studied by biochemical and cytochemical methods. Both parts of the study were performed on isolated open pig thyroid follicles in which the cells have preserved polarity and both the apical and basal cell surfaces are exposed to the incubation medium. The biochemical studies, performed with the scopoletin fluorescence assay, showed that H2O2 was released from the follicles into the medium at a rate of 0.5-1.0 nmol min-1 mg-1 DNA under basal conditions. The H2O2 release rate was promptly increased about 10 times by addition of the ionophore A23187 to Ca2+-containing medium. TSH caused an acute but weaker stimulation of H2O2 release, whereas (Bu)2cAMP was without effect, indicating that the TSH action was linked to Ca2+. Both basal and stimulated H2O2 release were strongly inhibited by p-chloromercuribenzene sulfonate. The cytochemical study, performed with the cerium technique, confirmed our previous observations on rat thyroid follicles. Reaction product was found on the apical cell surface but never on the basal cell surface or intracellularly. The apical reaction was enhanced by NADH and NADPH as well as by A23187 in Ca2+-containing medium. The apical reaction was strongly inhibited by p-chloromercuribenzene sulfonate. The observations indicate that the H2O2 released from the open follicles is generated on the apical plasma membrane of the follicle cells, possibly by NAD(P)H oxidase in this membrane. Furthermore, Ca2+ seems to be an important factor in the regulation of this H2O2 generation and, through that, in the regulation of iodination.

Predominance of alpha 2-adrenoceptors in porcine thyroid: biochemical and pharmacological correlations.

alpha-Adrenergic agonists such as norepinephrine and clonidine, but not methoxamine, inhibited the increase in cAMP accumulation elicited by TSH in porcine thyroid slices. The inhibitory effect of norepinephrine was abolished by yohimbine but not by prazosin. Likewise, the in vitro release of T4 from the pig thyroid slice stimulated by TSH was inhibited by norepinephrine and the inhibition was antagonized by yohimbine but not by prazosin. These results suggest that the inhibitory effect of alpha-adrenergic agonists is mediated through alpha 2-adrenoceptors. Preincubation of the thyroid slices with islet-activating protein, pertussis toxin, decreased the inhibitory effect of norepinephrine on the cAMP content, suggesting the involvement of guanine nucleotide regulatory protein (Ni) in the inhibition mediated through alpha 2-adrenoceptors. Binding studies with [3H]yohimbine and [3H]prazosin to porcine thyroid membranes revealed the specific and saturable binding of both radioligands. Scatchard analysis of the specific binding revealed a single class of binding sites for each radioligand; however, the maximum number of binding sites for [3H]yohimbine was about 10 times more than that for [3H]prazosin. In accordance with the pharmacological results, binding studies showed a predominance of alpha 2-adrenoceptors in the pig thyroid.

In vitro generation of NADPH as an index of thyroid stimulating immunoglobulins (TGI) in goitrous disease.

A quantitative cytochemical assay for the measurement of total NADPH formation in guinea pig thyroid tissue following incubation with thyroid stimulators was validated and applied to the measurement of such stimulators in IgG concentrates prepared from human plasma. Pentose shunt enzyme activity was not uniformly distributed in thyroid tissue but this could be overcome and NADPH generation in such tissue used to assess accurately thyroid stimulators if a sufficient number of thyroid cells were measured. Specificity studies showed that antiserum to human IgG significantly diminished the NADPH generating capacity of IgG concentrates prepared in plasma from goitrous patients while antiserum to h-TSH had no such effect. The measurement of thyroid growth stimulating immunoglobulins (TGI) depended not only on the amount of NADPH generated but also on the IgG concentration at which maximum responses occurred. TGI present in goitrous Graves' disease were 10 times more potent than those present in euthyroid goitre, while such stimulators when present in toxic nodular goitre appeared to possess an intermediate potency. This finding, when taken together with the demonstration that TSH stimulation decreased intercellular differences in pentose shunt activity, provides experimental evidence for the hypothesis that an acute intense growth stimulus affecting all thyroid cells produces the diffuse hyperplasia characteristic of Graves' disease. In contrast, the weaker stimulus observed in nodular goitre may, over a period of time, result in the asymmetric hyperplasia commonly observed in this condition.

A structural comparison of guinea pig thyroid and fat TSH receptors by photoaffinity labelling

TSH receptors from guinea pig thyroid and epididymal fat have been covalently crosslinked to 125I-labelled TSH conjugated to N-hydroxysuccinimidyl-4-azidobenzoate. Analysis by SDS—PAGE and autoradiography showed bands corresponding to TSH subunits ( M r 14 000) and intact TSH ( M r 28 000; subunits crosslinked) and two at higher M r separated by 14 000. The latter bands represented one or two subunits of TSH crosslinked to a subunit of the TSH receptor with M r 57 000 (fat) or 60 000 (thyroid). These M r values were reduced by trypsin treatment to 43 000 and 50 000, respectively. Analysis under non-reducing conditions showed that both fat and thyroid receptors have a second disulphide linked subunit of M r 30 000.

Immunocytochemical study on the C cells in pig thyroid glands.

The distribution of pig thyroid C cells was demonstrated by a immunoperoxidase method using antiserum to porcine calcitonin. The average number of C cells per 4 mm2 counted in the deep and superficial regions was 127 and 55, respectively. The number in the cranial, middle and caudal regions was 60, 128 and 83, respectively. The number of C cells per follicular and C cells (C cell concentration) in deep and superficial regions was 4.9 and 2.6%. It was 3.2, 4.9 and 3.3% in the cranial, middle and caudal regions, respectively. These results indicate that the number and concentration of C cells were not only larger in deep than superficial regions but also in the middle region than in cranial and caudal in pig thyroid gland. These results also indicate that the C cell distribution in pig thyroid gland is the same as that of dog, human, cat, rat, guinea pig, and mouse.

Intracellular distribution of AMP deaminase in the pig thyroid gland.

AMP deaminase and adenosine deaminase activities were assayed in the subcellular fractions of pig thyroid gland. AMP deaminase is localized in the cytosolic fraction; however, this enzyme showed remarkable tendency to bind with the subcellular particulate fractions. Adenosine deaminase is also localized in the cytosolic fraction but in contradistinction to AMP deaminase, adenosine deaminase under the same experimental conditions has no tendency of binding to subcellular particulate fractions. The significance of AMP deaminase binding to subcellular particulate fractions is discussed.

TRH immunoreactivity in the thyroid gland.

Methanol extracts of dog and pig thyroid tissue contained considerable amounts of thyrotropin releasing hormone immunoreactivity (iTRH): 2.98 +/- 1.38 and 1.83 +/- 0.58 pmol/g thyroid wet weight, respectively (mean +/- SD). This iTRH was not due to ether extractable materials and behaved like TRH standard in dilution experiments, during gel filtration on Sephadex G-25 and during thin layer chromatography on a silica gel plate. Cation exchange chromatography on SP-Sephadex C-25 revealed that the iTRH in methanol extract was heterogeneous. However, part of the iTRH applied on the column was eluted as synthetic TRH: 0.209 +/- 0.046 and 0.052 +/- 0.021 pmol/g dog and pig thyroid, respectively. During incubation with rat serum, the SP-Sephadex purified iTRH disappeared in parallel with TRH. These studies indicate that the thyroid contains small amounts of iTRH which behaves identically to synthetic TRH.

Ontogeny of immunoreactive somatostatin in thyroid C cells from dogs and guniea pigs.

The development of immunoreactive somatostatin in thyroid C cells of dogs and guinea pigs from early fetuses to adults was investigated by the use of immunoperoxidase histochemistry and radioimmunoassay. The time of appearance and developmental patterns of immunoreactive somatostatin in the C cells were completely different in both species. In guinea pig thyroids, the somatostatin immunoreactivity appeared later than the calcitonin immunoreactivity and the number of somatostatin-positive cells was very small during fetal periods. The somatostatin immunoreactivity rapidly increased during neonatal periods. A large population of the somatostatin cells and a high concentration of somatostatin immunoreactivity were observed in mature animals. On the other hand, in dog fetuses somatostatin immunoreactivity appeared very early, at the same time as the calcitonin immunoreactivity. The largest population of somatostatin cells was found at the stage when the primordial follicles were vigorously formed throughout whole thyroid parenchyma. At this stage almost all of calcitonin-positive cells were also somatostatin-positive. The somatostatin cells progressively decreased as the development proceeded, in contrast to the calcitonin cells which increased with gestational age. In postnatal dogs only a few C cells revealed the immunoreaction for somatostatin, and the concentration of somatostatin was very low. These findings suggest that the function of somatostatin in dog thyroid C cells may be different from that in guinea pig C cells.

Defective replication of porcine transmissible gastroenteritis virus in a continuous cell line.

During a search for established cell lines to produce large quantities of porcine transmissible gastroenteritis virus (TGEV), we observed bright immunofluorescent staining 6- 12h after infection of pig kidney derived LLC-PK1 line. Infectious virus yield was, however, 2 log10 lower than that from secondary adult pig thyroid (APT/2) cell cultures, although small plaques were visible by three days in cultures maintained under agarose, suggesting limited replication. Attempts to adapt TGEV to the LLC-PK1 cell line by 10 serial 20h passes were unsuccessful. Procedures to purify virions from infected LLC-PK1 cells produced less than 1% of the particles isolated from parallel APT/2 cultures. Examination of intracellular viral RNA in actinomycin-D treated cells revealed similar amounts of genomic RNA and the 4 major subgenomic species in both cell types, suggesting that there was no defect in viral RNA replication. In vitro translation of polyadenylated RNA from infected APT/2 and LLC-PK1 cells, followed by immune precipitation of the products, showed similar profiles of precursors to structural polypeptides, confirming the functional integrity of the viral messengers in the restrictive cell. Comparison of the viral polypeptides synthesised following infection of the two cell types showed that similar species were synthesised in both, corresponding to a group of 28-30,000 mol. wt. envelope glycopolypeptides, a 47,000 mol. wt. nucleoprotein and peplomer glycopolypeptides of about 200,000 mol. wt. The rate of viral polypeptide synthesis in LLC-PK1 cells was reproducibly higher than in APT/2, resulting in the earlier detection of bands and greater incorporation of isotope. Tunicamycin at 1 microgram/ml had a similar effect in both cells, preventing glycosylation of the 26,000 mol. wt. precursor of the envelope glycopolypeptides and synthesis of the 200,000 peplomer glycoprotein. Degradation of the nucleoprotein from 47,000 to 42,000 mol. wt. although detectable in both cells was more marked in the LLC-PK1 cultures. Phosphorylation of these proteins was readily demonstrated in both cells, although phosphorylation of host proteins and, to some extent, viral envelope proteins was considerably greater in the LLC-PK1. The significance of this finding with respect to virus maturation is being investigated.

Transcytosis in thyroid follicle cells.

Inside-out follicles prepared from pig thyroid glands were used for studies on endocytosis. endocytosis. In this in vitro system, only the apical plasma membranes of follicle cells were exposed to tracers added to the culture medium. Cationized ferritin (CF) bound to the apical plasma membrane and was transferred first to endosomes and to lysosomes (within 5 min). Later, after approximately 30 min, CF was also found in stacked Golgi cisternae. In addition, a small fraction of endocytic vesicles carrying CF particles became inserted into the lateral (at approximately 11 min) and the basal (at approximately 16 min) plasma membranes. Morphometric evaluation of CF adhering to the basolateral cell surfaces showed that the vesicular transport across thyroid follicle cells (transcytosis) was temperature-sensitive; it ceased at 15 degrees C but increased about ninefold in follicles stimulated with thyrotropin (TSH). Thyroglobulin-gold conjugates and [3H]thyroglobulin (synthesized in separate follicle preparations in the presence of [3H]leucine) were absorbed to the apical plasma membrane and detected mainly in lysosomes. A small fraction was also transported to the basolateral cell surfaces where the thyroglobulin preparations detached and accumulated in the newly formed central cavity. As in the case of CF, transcytosis of thyroglobulin depended on the stimulation of follicles with TSH. The observations showed that a transepithelial vesicular transport operates in thyroid follicle cells. This transport is regulated by TSH and includes the transfer of thyroglobulin from the apical to the basolateral plasma membranes. Transcytosis of thyroglobulin could explain the occurrence of intact thyroglobulin in the circulation of man and several mammalian species.

Lysosomal membrane recovery following labilization by thyroid stimulators.

Lysosomal membrane permeability was assessed by measuring freely available naphthylamidase activity in intact preparations of guinea pig thyroid follicular cells following exposure of thyroid tissue to sequential stimulation by two thyroid stimulators, thyrotrophin (TSH) and thyroid stimulating immunoglobulins (TSI). These investigations showed that following labilization by TSH, the lysosomal membranes recovered and were capable of responding to a second thyroid stimulator (TSI). That such recovery represented restabilization of lysosomal membranes was confirmed by the finding that latent naphthylamidase activity was restored without a change in total activity of the enzyme.

Control of glycoprotein synthesis.

Hen oviduct membranes have been shown to catalyze the transfer of GlcNAc from UDP-GlcNAc to GlcNAc-beta 1-2Man alpha 1-6(GlcNAc beta 1-2 Man alpha 1-3) Man beta 1-4GlcNAc beta 1-4GlcNAc-Asn-X (GnGn) to form the triantennary structure GlcNAc beta 1-2Man alpha 1-6[GlcNAc beta 1-2(GlcNAc beta 1-4)Man alpha 1-3]Man beta 1-4GlcNAc beta 1-4GlcNAc-Asn-X. The enzyme has been named UDP-GlcNAc:GnGn (GlcNAc to Man alpha 1-3) beta 4-N-acetylglucosaminyltransferase IV (GlcNAc-transferase IV) to distinguish it from three other hen oviduct GlcNAc-transferases designated I, II, and III. Since GlcNAc-transferases III and IV both act on the same substrate, concanavalin A/Sepharose was used to separate the products of the two enzymes. At pH 7.0 and at a Triton X-100 concentration of 0.125% (v/v), GlcNAc-transferase IV activity in hen oviduct membranes is 7 nmol/mg of protein/h. The product was characterized by high resolution proton NMR spectroscopy at 360 MHz and by methylation analysis. In addition to triantennary oligosaccharide, hen oviduct membranes produced about 20% of bisected triantennary material, GlcNAc beta 1-2Man alpha 1-6[GlcNAc beta 1-2(GlcNAc beta 1-4)Man alpha 1-3] [GlcNAc beta 1-4]Man beta 1-4GlcNAc beta 1-4GlcNAc-Asn-X. Maximal GlcNAc-transferase IV activity requires the presence of both terminal beta 1-2-linked GlcNAc residues in the substrate. Removal of the GlcNAc residue on the Man alpha 1-6 arm or of both GlcNAc residues reduces activity by at least 80%. A Gal beta 1-4GlcNAc disaccharide on the Man alpha 1-6 arm reduces activity by 68% while the presence of this disaccharide on the Man alpha 1-3 arm reduces activity to negligible levels. A similar substrate specificity was found for GlcNAc-transferase III, the enzyme which adds a bisecting GlcNAc in beta 1-4 linkage to the beta-linked Man residue. Since a bisecting GlcNAc was found to prevent GlcNAc-transferase IV action, the bisected triantennary material found in the incubation must have been formed by the sequential action of GlcNAc-transferase IV followed by GlcNAc-transferase III. Activities similar to GlcNAc-transferase IV were also detected in rat liver Golgi-rich membranes (0.4 nmol/mg/h) and pig thyroid microsomes (0.1 nmol/mg/h).

Purification and characterization fo a phosvitin kinase from the thyroid gland

1. Two cyclic AMP independent protein kinases phosphorylating preferentially acidic substrates have been identified in soluble extract from human, rat and pig thyroid glands/ Both enzymes were retained on DEAE-cellulose. The first enzyme activity eluted between 60 and 100 mM phosphate (depending on the species), phosphorylated both casein and phosvitin and was retained on phosphocellulose; this enzyme likely corresponds to a casein kinase already described in many tissues. The second enzyme activity eluted from DEAE-cellulose at phosphate concentrations higher than 3000 mM, phosphorylated only phosvitin and was not retained on phophocellulose. These enzymes were neither stimulated by cyclic AMP, cyclic GMP and calcium, nor inhbiited by the inhibitor of the cyclic AMP dependent protein kinases. 2. The second enzyme activity was purified from pig thyroid gland by the association of affinity chromatography on insolubilized phosvitin and DEAE-cellulose chromatography. Its specific activity was increased by 8400. 3. The purified enzyme (phosvitin kinase) was analyzed for biochemical and enzymatic properties. Phosvitin kinase phosphorylated phosvitin with an apparent K m of 100 μg/ml; casein, histone, protamine and bovine serum albumin were not phosphorylated. The enzyme utilized ATP as well as GTP as phosphate donor with an apparent K m of 25 and 28 μM, respectively. It had an absolute requirement for Mg 2+ with a maximal activity at 4 mM and exhibited an optimal activity at pH 7.0. The molecular weight of the native enzyme was 110 000 as determined by Sephacryl S300 gel filtration. The analysis by SDS-polyacrylamide gel electrophoresis revealed a major band with a molecualr weight of 35 000 suggesting a polymeric structure of the enzyme.

39] Preparation of inside-out thyroid follicles for studies on transcellular transport (transcytosis)

This chapter describes the preparation of inside-out thyroid follicles for studies on transcellular transport also referred to as “transcytosis.” Studies on the mechanism of transepithelial passage of thyroglobulin can be facilitated by using an in vitro system where access to the apical plasma membrane is possible. Such a system is provided by preparations of inside-out follicles from pig thyroid gland. The chapter describes the conditions that lead to the formation of inside-out follicles and allows studying the transepithelial vesicular traffic from the apical to the basolateral cell surfaces. Collagenase digestion of thyroid tissue and application of shearing forces by pipetting result in the dissociation of tissue into intact segments of follicles opened during pipetting. Fibroblasts and parafollicular and endothelial cells are separated from follicle segments by low-speed differential centrifugation. Purified follicle segments form closed follicular spheres with a reversed polarity within 24 hr of suspension culture. The polarity of all follicles in suspension is reversed so that the apical plasma membrane is oriented toward the culture medium, and the basal cell surface is directed toward the central cavity.

Binding of 125I-human TSH by gamma globulins of sera containing thyroid-stimulating immunoglobulin (TSI)

Anti-IgG precipitable 125I-hTSH binding activity of TSI-containing serum is increased as compared to normal serum after adsorption with pig thyroid membranes. This IgG binding of 125I-hTSH can be inhibited with nonradioactive TSH. We have hypothesized that TSI may be an antiidiotypic antibody to anti-TSH. This evidence for the presence of anti-TSH in TSI-containing sera supports the hypothesis.

Effects of eicosatetraynoic acid (ETYA) on cultured pig thyroid cells: Relationships between the inhibition of the phosphatidate—phosphatidyl inositol cycle, the iodination and the cyclic AMP responsiveness to thyrotropin

The arachidonate inhibition of the adenylate-cyclase system of cultured pig thyroid cells was not mediated by cyclooxygenase, lipoxygenase or peroxidase metabolites. Indeed ETYA, an inhibitor of cyclooxygenase and lipoxygenase, and methimazole, an inhibitor of peroxidase and iodination were without effect on the arachidonate inhibition. Moreover the effect of arachidonate was amplified by a combination with ETYA. In 32P incorporation experiments we observed a modification of the labelling of individual phospholipids of cultured pig thyroid cells resulting in a decrease into phosphatidylinositol (PI) and an increase into phosphatidate (PA) of arachidonate and ETYA-treated cells. These results may be explained by an inhibition of CDP-diacylglycerol: inositol transferase and conversely a stimulation of PI specific phospholipase C yielding a decrease in PI and an increase in PA, which inhibits in turn adenylate cyclase activity possibly by Ca 2+ translocation.