Serotonin as a homeostatic regulator of lactation.

Serotonin (5-HT), a neurotransmitter produced in mammary epithelial cells (MECs), acts via autocrine-paracrine mechanisms on MECs to regulate milk secretion in a variety of species. Recent studies in dairy cows reported that 5-HT ligands affect milk yield and composition. We determined the mRNA expression of bovine 5-HT receptor (5-HTR) subtypes in bovine mammary tissue (BMT) and cultured bovine MECs. We then used pharmacologic agents to evaluate functional activities of 5-HTR subtypes. The mRNAs for five receptor isoforms (5-HTR1B, 5-HTR2A, 5-HTR2B, 5-HTR4, and 5-HTR7) were identified by conventional reverse transcription PCR, real-time PCR, and in situ hybridization in BMT. In addition to luminal MEC expression, 5-HTR4 was expressed in myoepithelium, and 5-HTR1B, HTR2A, and HTR2B were expressed in small mammary blood vessels. Studies to date report that there are multiple 5-HTR isoforms in mammary tissue of rodents, humans, and cattle. Inhibition of the 5-HT reuptake transporter with selective 5-HT reuptake inhibitors (SSRIs) disrupted tight junctions and decreased milk protein mRNA expression in mouse, human, and bovine mammary cells. Selective 5-HT reuptake inhibitors act to increase the cellular exposure to 5-HT by preventing reuptake of 5-HT by the cell and eventual degradation. Increasing 5-HT concentration in milk via inhibiting its reuptake (SSRI), or by increasing the precursor for 5-HT synthesis 5-hydroxytryptophan, accelerated decline in milk synthesis at dry-off. We conclude that the 5-HT system in mammary tissue acts as a homeostatic regulator of lactation.

Suppression of lactation and acceleration of involution in the bovine mammary gland by a selective serotonin reuptake inhibitor.

Serotonin (5-HT) is a homeostatic regulator of lactation. Selective 5-HT reuptake inhibitors (SSRI) are commonly prescribed pharmaceuticals that inhibit activity of the 5-HT reuptake transporter, increasing cellular exposure to 5-HT. Use of SSRIs has been shown to alter lactation performance in humans and 5-HT has been shown to reduce milk yield in cattle. However, it has not been determined how SSRI treatments affect the bovine mammary gland. We evaluated the effects of SSRI (fluoxetine (FLX)) administration on tight junctions (TJs) and milk protein gene expression in a lactogenic culture model, using primary bovine mammary epithelial cells (pBMEC). Additionally, we evaluated the effects of intramammary infusions of FLX and 5-hydroxytryptophan on milk production and TJ status in multiparous Holstein cows at dry-off. Treatment of pBMEC cultured on permeable membranes disrupted TJs, as measured by transepithelial resistance and immunostaining for zona occludens 1. Correspondingly, treatment of '3D', collagen-embedded lactogenic cultures of pBMEC with FLX suppressed milk protein gene expression (α-lactalbumin and ß-casein) in a concentration-dependent manner. Finally, intramammary treatment of Holstein cows with FLX resulted in an accelerated rate of milk decline. Additionally, TJ permeability increased in FLX-treated animals, as measured by plasma lactose and milk Na(+) and K(+) levels. Results of these experiments imply that SSRI administration accelerates the rate of mammary gland involution through disassembly of TJs and inhibition of milk protein gene expression in vitro and in vivo, leading to reduction of milk yield.

Serum levels of prolactin, growth hormone, and cortisol in burn patients: correlations with severity of burn, serum cytokine levels, and fatality.

In this study, we measured serum prolactin (PRL), cortisol, growth hormone, interleukin (IL)-1beta, IL-6, IL-8, IL-10, IL-12, and tumor necrosis factor-alpha in patients admitted with small-to-moderate burn injuries. Serum samples were obtained at the time of admission from 49 adult male burn patients with ages ranging from 18 to 91 years and TBSA ranging from 0.001 to 60%. The levels of serum PRL, IL-8, IL-6, and IL-1beta correlated positively with the TBSA, whereas only serum IL-8 levels correlated positively with fatality. Each of these factors were increased at least 2-fold at the higher burn severity. Not surprisingly, there was a large degree of variability in the hormone and cytokine levels in this patient population, which presumably reflects individual levels of stress, as well as other physiological variables. We also studied relationships between serum hormone levels and serum cytokine levels in this context. Linear regression analysis revealed a significant positive correlation between the serum PRL level and the levels of IL-10, IL-6, and IL-8. These results indicate that PRL responds to burn injury at early time points and that a subset of cytokines are involved in the early response to burn injury.

Stress hormone secretion and gut signal transducer (STAT) proteins after burn injury in rats.

A burn injury triggers traumatic reactions characteristics of a stress. Here we investigated the early responses of prolactin (PRL), corticosterone (CS), and signal transducer and activator of transcription 5 (STAT5) in male Sprague-Dawley rats after burn injury. PRL and CS levels were determined in blood serum. STAT5 and phospho-STAT5 levels were determined in jejunum total protein extracts. The results confirmed an expected increase of CS between 4 and 6 h after the burn injury. Unexpectedly, PRL secretion was suppressed during the same time frame. These hormone levels returned to normal 6 to 8 h after burn injury. STAT5 was increased in the jejunum after burn injury, and its phosphorylation was increased between 8 and 11 h after burn injury. These changes in STAT5 were not temporally correlated with either the hormone changes that we observed or with previously documented changes of the gut function after burns.

Anterior pituitary hormones, stress, and immune system homeostasis.

An extensive, and controversial, literature concluding that prolactin (PRL), growth hormone (GH), insulin-like growth factor-I (IGF-I), and thyroid hormones are critical immunoregulatory factors has accumulated. However, recent studies of mice deficient in the production of these hormones or expression of their receptors indicate that there are only a few instances in which these hormones are required for lymphocyte development or antigen responsiveness. Instead, a case is made that their primary role is to counteract the effects of negative immunoregulatory factors, such as glucocorticoids, which are produced when the organism is subjected to major stressors. The immunoprotective actions of PRL, GH, IGF-I, and/or thyroid hormones in these instances may ensure immune system homeostasis and reduce the susceptibility to stress-induced disease. These immuno-enhancing effects could be exploited clinically in instances where the immune system is depressed due to illness or various treatment regimens.

Glycosylation-dependent cell adhesion molecule 1 (GlyCAM 1) is induced by prolactin and suppressed by progesterone in mammary epithelium.

Glycosylation-dependent cell adhesion molecule 1 (GlyCAM 1), a mucin-like endothelial glycoprotein, was induced by PRL and suppressed by progesterone in the mammary gland of mice, and in HC11 mouse mammary epithelial cells. Complementary DNA microarray analysis revealed that expression of GlyCAM 1 was reduced in the mammary gland of PRL-gene disrupted mice (PRL-/-) compared with control (PRL+/-) littermates. This result was confirmed by in situ hybridization and immunostaining. The messenger RNA (mRNA) encoding GlyCAM 1 was present in mammary epithelia of PRL-stimulated mice. Immunohistochemistry indicated that GlyCAM 1 protein was detectable both in mammary epithelia and in the ductal lumen in PRL+/- virgin mice, but not in PRL-/- mice. GlyCAM 1 mRNA was highly induced by grafting pituitary glands from normal littermates. Trace amounts of mRNA for GlyCAM 1 were detected by RT-PCR in mammary tissue of PRL-/- mice. Progesterone inhibited both basal and PRL-stimulated GlyCAM 1 transcription. In HC11 cells, GlyCAM 1 mRNA was induced in cells treated with insulin, dexamethasone, and PRL. Similar to the in vivo studies, progesterone inhibited the induction of GlyCAM 1 transcription. In CHO cells, PRL stimulated transcription of a luciferase reporter gene containing an 800-bp promoter fragment of GlyCAM 1, and progesterone partially suppressed the PRL effect. These data demonstrate that expression of GlyCAM 1 in mammary gland is under the control of both PRL and progesterone.

Prolactin gene disruption does not compromise differentiation of tuberoinfundibular dopaminergic neurons.

In mice with spontaneous mutations in transcription factors Prop-1 or Pit-1, the pituitary fails to produce prolactin (PRL), GH and TSH, and numbers of hypothalamic PRL-regulating dopaminergic (DA) neurons (area A12) are reduced by more than 50%. A normal neuronal population can be maintained in these mutants by PRL treatment of neonates, but not of adults. Targeted disruption of the PRL structural gene in mice provides a new model of isolated PRL deficiency to test the specificity of the PRL neurotrophic effect. The present study used morphological methods to assess hypophysiotropic tuberoinfundibular dopaminergic (TIDA) neurons in these mice, with the hypothesis that isolated PRL deficiency also would lead to reduction in TIDA neuron number. Brains of female and male homozygous PRL-null (- or -) mice and normal heterozygous (+ or -) siblings were compared using formaldehyde-induced endogenous catecholamine fluorescence and tyrosine hydroxylase (TH) immunocytochemistry. Immunostaining intensity was quantified using computerized image analysis, and total numbers of TH-immunoreactive neurons were counted in three diencephalic DA brain regions. Intensity of DA fluorescence in A12 perikarya and median eminence (ME) was reduced in - or - mice; fluorescence in other brain areas was comparable for - or - and + or - mice. Immunostaining intensity of TH was significantly lower (p = 0.0001) in - or - than in normal mice in perikarya of A12, but not in cell bodies of nonhypophysiotropic area A13 (medial zona incerta). In external ME, TH immunostaining intensity was lower (p = 0.0001) in PRL-null than in normal mice. The decrease in TH intensity in both perikarya and in ME was significant for both female and male - or - mice. However, numbers of A12 neurons in the PRL-null mice were not lower than those of normal siblings. TH-immunoreactive cell number also did not differ between + or - and - or - mice in areas A13 and periventricular A14. The presence of a normal complement of A12 DA neurons in the PRL-null mice, despite greatly reduced DA and TH, emphasizes that steady-state content and differentiation of phenotype in individual neurons are very different assessments. The results suggest that, although absence of the stimulatory PRL feedback signal results in diminished activity of TIDA neurons, differentiation of these cells is not adversely affected.

The roles of prolactin, growth hormone, insulin-like growth factor-I, and thyroid hormones in lymphocyte development and function: insights from genetic models of hormone and hormone receptor deficiency.

An extensive literature suggesting that PRL, GH, IGF-I, and thyroid hormones play an important role in immunity has evolved. Because the use of one or more of these hormones as immunostimulants in humans is being considered, it is of critical importance to resolve their precise role in immunity. This review addresses new experimental evidence from analysis of lymphocyte development and function in mice with genetic defects in expression of these hormones or their receptors that calls into question the presumed role played by some of these hormones and reveals unexpected effects of others. These recent findings from the mutant mouse models are integrated and placed in context of the wider literature on endocrine-immune system interactions. The hypothesis that will be developed is that, with the exception of a role for thyroid hormones in B cell development, PRL, GH, and IGF-I are not obligate immunoregulators. Instead, they apparently act as anabolic and stress-modulating hormones in most cells, including those of the immune system.

Interleukin-6 signal transduction in human intestinal epithelial cells.

The gut is an important source of inflammatory cytokines, but there is scant information on the mechanisms of cytokine action in gut epithelium. We hypothesized that in human Caco-2 cells, IL-6 acts directly through stimulation of Stat phosphorylation and that bacterial lipopolysaccharide (LPS) causes Stat activation indirectly because of its ability to cause the autocrine secretion and action of interleukin (IL)-6. Stat1, Stat5a, and Stat5b, but not Stat3, were detected in Caco-2 cells. DNA-binding activity corresponding to activated Stat5 was stimulated in a biphasic manner by IL-6, with a transient early phase, followed by sustained activation between 8 and 48 h. LPS also stimulated Stat5-like binding, but there was no early phase of activation. Functional tests of Stat5 activation showed that IL-6 stimulated Stat5-dependent reporter gene transcription but had no effect on Stat1-dependent transcription. LPS did not stimulate Stat-dependent transcription, nor did it alter the transcriptional response to IL-6. Tyrosine phosphorylation of both Stat5a and Stat5b was induced by IL-6. We infer from these data that IL-6 acts on intestinal epithelia through a Stat5-mediated transcriptional mechanism, whereas LPS does not induce gene expression through autocrine activation of enterocyte Stat signaling. These data provide a basis for testing the in vivo regulation of gut signaling and the interaction of gut reticuloendothelial cells with epithelial signal transduction.

Prolactin gene-disruption arrests mammary gland development and retards T-antigen-induced tumor growth.

Prolactin (PRL), interacting with other hormones from the pituitary, gonad, and placenta, activates specific signals that drive the appropriately timed morphological and functional development of the mammary gland. A mouse model of isolated PRL deficiency (PRL-/-) was created by gene disruption in an effort to further understand the molecular basis of mammary gland development and breast cancer. Whereas primary ductal growth was normal in PRL-/- mice, ductal arborization was minimal (branches/mm2=1.5+/-0.5), and lobular budding was absent. Replacement therapy with PRL injections stimulated a modest degree of lobular budding and ductal arborization (3.75+/-0.9). Pituitary transplants to the kidney capsule of PRL-/- mice restored lobular budding and ductal arborization, to the full extent of that seen in control animals (20. 3+/-5.5). Pregnancy, established by mating progesterone-treated PRL-/- females with PRL-/- males, led to complete morphological development of the mammary gland, appropriate to the gestational stage. PRL treatment stimulated tyrosine phosphorylation and DNA binding activity of Stat5a, but not Stat1 in PRL-/- or PRL+/- females, and Stat5a, but not Stat1, was elevated by estradiol within 24 h. PRL-deficient mice were crossed with mice expressing a dominant oncogene (polyoma middle-T antigen driven by the MMTV promoter, PyVT mice). Palpable (1 mm3) tumors were detected an average of 9 days earlier in hormonally normal females (PRL+/-:PyVT) compared with littermates that were PRL-deficient (PRL-/-:PyVT). The growth rate of PyVT-induced tumors was 30% faster in PRL+/-, than in PRL-/- females.

Prolactin-independent modulation of the beta-casein response element by Erk2 MAP kinase.

The MAP kinases have been suggested to play a role in intracellular signalling by PRL. A reporter gene construct, PRE3-CAT, which manifests PRL responsiveness through a Stat5-binding site (PRE), was induced by PRL in CHO cells expressing the PRL-R. A fusion protein (Gal4-Stat5(695)), containing the C-terminal domain of Stat5a (amino acids 695-794) linked to the DNA-binding domain of Gal4 (Gal4 DBD), strongly activated transcription of a luciferase reporter gene. Therefore, the Stat5 C-terminus, which contains a potential MAP kinase phosphorylation site, exhibits a modular transactivating function. A kinase-defective mutant of Erk2 (iMAPK) caused a dose-dependent suppression of PRL-stimulated PRE3-CAT, and also inhibited the induction of PRE3-CAT by Jak2 over-expression. Correspondingly, over-expression of the MAP kinase activator v-Src increased the PRL-stimulated level of PRE3-CAT. Gal4-Stat5(695) activity was not modulated by PRL or Jak2, consistent with the absence of the relevant tyrosine phosphorylation site at residue 694. Gal4-Stat5(695) was not inhibited by iMAPK, indicating that the C-terminal transactivation region of Stat5a is not sensitive to direct modulation of a MAP kinase pathway. These results suggest that alteration of Erk2 activity by growth factors may modulate PRL-induced gene expression by a mechanism upstream of Stat5.

Prolactin and mammary gland development.

Prolactin (PRL) regulates the development of the mammary gland at three stages in the reproductive life history of females. The first stage is mammary gland organogenesis, during which PRL contributes to the maturation of the mammary glands from a primary ductal system, which grows from terminal end buds, to the fully mature nonpregnant gland. The mature mammary gland is characterized by an absence of terminal end buds, and the development of a highly branched architecture, which is decorated by lobular buds. During pregnancy PRL, placental lactogens, and progesterone stimulate the expansion and physiological differentiation of the lobuloalveolar system from the lobular buds. After delivery PRL, in the context of falling progesterone, stimulates the final induction of milk protein gene expression and lactation. PRL acts directly on the mammary epithelium, and indirectly by stimulating luteal progesterone secretion in rodents. Disruption of the genes for PRL and the PRL receptor, as well as those for transcription factors important in mammary gland regulation (Stat proteins), have provided a new set of animal models with which to study normal mammary gland development and the relationships of PRL to breast carcinogenesis. Two major deficiencies in our current knowledge of PRL actions are our understanding of the role of epithelial-stromal interactions in PRL-induced mammary morphogenesis, and the identity of developmentally important genes that are regulated by PRL during normal mammary gland organogenesis.

Neuroendocrine and reproductive functions in male mice with targeted disruption of the prolactin gene.

Mice with a targeted disruption (knock-out) of the PRL gene (PRL-KO) were used to study the physiological role of PRL in the control of male neuroendocrine functions related to reproduction. Compared with normal males, PRL-KO mice had significant reductions in median eminence dopamine content, plasma LH levels, LH and FSH secretion in vitro (per mg pituitary), and weights of seminal vesicles and ventral prostate. PRL was not detectable in incubation medium with pituitaries from PRL-KO mice. No alterations were detected in PRL-KO mice in median eminence norepinephrine, plasma testosterone levels, or testosterone release (per mg testis) in vitro with or without LH. No differences were detected in PRL-KO vs. normal male mice in the interval from housing with normal female mice until conception, rate of pregnancy, or the number of live pups per litter. Pituitary weight in PRL-KO mice was increased (1.78 +/- 0.22 vs. 3.35 +/- 0.20 mg; P < 0.001), presumably due to reduced feedback inhibition and hypertrophy and/or hyperplasia of nonfunctional lactotrophs. These results indicate that the absence of PRL reduces pituitary LH release, attenuates median eminence dopaminergic activity, and affects the growth of seminal vesicles and ventral prostate. Although it was previously shown that PRL can repair the reproductive defect in male pituitary dwarf mice, our current results imply that the PRL deficiency alone is not sufficient to cause male infertility, although there are obvious alterations in reproductive neuroendocrine function in PRL-KO males.

Identification of two Y-box binding proteins that interact with the promoters of columbid annexin I genes.

Two annexin I (anxI) genes, called cp35 and cp37, are expressed from the pigeon (Columba livia) genome, but they are regulated differently at both the transcriptional and post-transcriptional levels. The proximal promoter elements of these two genes are very similar. A conserved sequence from the cp35 and cp37 promoters bound specifically with proteins present in cropsac cell extracts. This sequence of DNA was used to screen a lambdagt11 cDNA expression library. Clones encoding two pigeon Y-box binding proteins (YB) were isolated. One of the pigeon YB cDNAs was found to be most similar to YB1 from other species, and the other was most similar to chicken YB2. Each YB is encoded by a single-copy gene in the pigeon, and their mRNAs are expressed in many tissues. On Northern blots, the sizes of the mRNAs encoding pigeon YB1 (pYB1) and pigeon YB2 (pYB2) were 1.8 and 1.7kb, respectively. The sequences of both pYB1 and pYB2 diverge from their previously identified relatives in the N-terminal domain 'A'. Antisera were developed to unique peptide epitopes in YB1 or 2. Affinity-purified anti-YB1 and anti-YB2 detected immunoreactive proteins in extracts from a variety of pigeon tissues, including the cropsac. To confirm that pYB1 and pYB2 interact with the cp35 promoter, electrophoretic gel mobility shift reactions were carried out in the presence or absence of YB antibodies. Binding to the cp35 promoter was specifically neutralized by either anti-pYB1 or anti-pYB2. These results are the first evidence that two YB proteins simultaneously bind to a promoter element, and thereby may interact during regulation of gene expression.

Prolactin receptor heterogeneity in bovine fetal and maternal tissues.

Study of diverse PRL actions at a variety of fetal and maternal targets during pregnancy is complicated by receptor heterogeneity and multiple ligands circulating at this time. In the present studies, we have examined PRL receptors at a variety of potential targets by reverse transcription-PCR and Western analysis. Bovine tissues contain two different transcripts for the PRL receptor; the one that encodes a short form includes an additional 39 bases at a position identical to the deviation from the long form found in rodents and sheep. Western analyses of PRL receptors in microsomal fractions from various maternal and fetal tissues revealed considerable size heterogeneity. Collectively, the larger immunoreactive moieties (apparent Mr 100 kDa or greater) and the smaller species (47-55 kDa) correlated well with the relative abundance of the transcripts for the different forms of the receptor and varied considerably among tissues. N-Glycosylation was shown to be the major, but not the only, modification of both receptor forms when transiently transfected into COS-7 and END-6.2 cells. Much of the short form could be reduced to the mobility predicted from the complementary DNA by culture with tunicamycin; this was not true of the long form, suggesting modifications specific for its cytoplasmic domain. Differences in the pattern of immunoreactive species in the COS-7 and END-6.2 cells are consistent with cell-specific modifications. The ability of these receptor forms to mediate a transcriptional response to PRL and its placental relative, placental lactogen, was evaluated with a PRL response element inserted upstream from a thymidine kinase promoter/reporter gene construct transiently transfected into CHO-K1 cells. Both hormones were able to stimulate reporter gene expression through the long form, but not the short form, of the receptor. These studies will facilitate examination of tissue-specific actions of PRL and related hormones during pregnancy.

The logic of signaling from the cell surface to the nucleus.

The apparent imprecisions in the signal transduction mechanisms that couple specific hormone receptors to predictable nuclear transcription events have raised many issues for both biology and medicine. In this article, a fuzzy logic model is proposed to provide a means of formally describing signal transduction systems and making predictions about the effects of various combinations of inputs. The fuzzy logic model may provide opportunities mathematically to describe regulatory ensembles that are too complex to describe by conventional models. In addition, the nature of "fuzzy variables" provides insight into the paradoxes that underlie both imprecision and predictability in signal transduction.

Defective mammopoiesis, but normal hematopoiesis, in mice with a targeted disruption of the prolactin gene.

Prolactin (PRL) has been implicated in numerous physiological and developmental processes. The mouse PRL gene was disrupted by homologous recombination. The mutation caused infertility in female mice, but did not prevent female mice from manifesting spontaneous maternal behaviors. PRL-deficient males were fertile and produced offspring with normal Mendelian gender and genotype ratios when they were mated with heterozygous females. Mammary glands of mutant female mice developed a normal ductal tree, but the ducts failed to develop lobular decorations, which is a characteristic of the normal virgin adult mammary gland. The potential effect of PRL gene disruption on antigen-independent primary hematopoiesis was assessed. The results of this analysis indicated that myelopoiesis and primary lymphopoiesis were unaltered in the mutant mice. Consistent with these observations in PRL mutant mice, PRL failed to correct the bone marrow B cell deficiency of Snell dwarf mice. These results argue that PRL does not play any indispensable role in primary lymphocyte development and homeostasis, or in myeloid differentiation. The PRL-/- mouse model provides a new research tool with which to resolve a variety of questions regarding the involvement of both endocrine and paracrine sources of PRL in reproduction, lactogenesis, tumorigenesis and immunoregulation.

Interactions among Janus kinases and the prolactin (PRL) receptor in the regulation of a PRL response element.

PRL regulates milk gene expression, at least in part, by activating JAK2 kinase and STAT5 (signal transducer and activator of transcription 5), initially termed mammary gland factor (MGF). These experiments were initiated to gain a better understanding of the mechanisms of transcriptional activation via PRL receptor (PRL-R) signaling. Binding of PRL to the recombinant pigeon PRL-R-activated transcription driven by a 2.8 kbp 5'-fragment of the rat beta-casein gene. PRL enhanced the expression of chimeric reporters containing the beta-casein PRL response element (PRE), but not the c-fos sis-inducible element, when the reporters were transfected into Chinese hamster ovary cells with the PRL-R. Wild type receptor, which contains a duplication of the entire extracellular ligand-binding domain, was only slightly more effective than a truncation mutant with a single extracellular domain. Transfection with either JAK1, JAK2, or JAK3 increased basal transcription through both the PRE and sis-inducible element. Coexpression of JAK2 with PRL-R resulted in amplification of the induction of the PRE by PRL, whereas JAKs 1 and 3 did not amplify the PRL effect. Overexpression of JAK2 mutants blocked PRE activation by PRL. Mutant JAK2 also interfered with PRE activation by JAK3 but did not affect JAK1's stimulatory effect.

Annexin VI overexpression targeted to heart alters cardiomyocyte function in transgenic mice.

Annexin VI is a member of a family of Ca(2+)-dependent phospholipid-binding proteins that is expressed in many tissues, including the heart. It is a regulator of membrane-associated events, including the skeletal muscle ryanodine-sensitive Ca2+ release channel and the cardiac Na+/Ca2+ exchanger. The potential roles of annexin VI in Ca2+ signaling in cardiac myocytes were evaluated by targeting its overexpression to the hearts of transgenic mice. Expression of full-length human annexin VI cDNA was targeted to the heart using the alpha-myosin heavy chain gene promoter (Subramaniam, A., W. K. Jones, J. Gulick, S. Wert, J. Neumann, and J. Robbins. J. Biol. Chem. 266: 24613-24620, 1991). Five transgenic lines exhibited at least 10-fold overexpression of annexin VI protein in both atria and ventricles. Pathological evaluation indicated mice overexpressing annexin VI had enlarged dilated hearts, acute diffuse myocarditis, lymphocytic infiltration, moderate to severe fibrosis throughout the heart, and mild fibrosis around the pulmonary veins of the lungs. Contractile mechanics of cardiomyocytes isolated from hearts of transgenic animals showed frequency-dependent reduced percent shortening and decreased rates of contraction and relaxation compared with control animals. Cardiomyocytes isolated from transgenic animals had lower basal levels of intracellular free Ca2+ and a reduced rise in free Ca2+ following depolarization. After stimulation, intracellular free Ca2+ returned to basal levels faster in transgenic cells than in cells from control animals. These data demonstrate that the overexpression of annexin VI in the heart disrupts normal Ca2+ homeostasis and suggests that this dysfunction may be due to annexin VI regulation of pumps and/or exchangers in the membranes of cardiomyocytes.