A variant beta-tubulin isoform of Drosophila melanogaster (beta 3) is expressed primarily in tissues of mesodermal origin in embryos and pupae, and is utilized in populations of transient microtubules

The beta 3-tubulin gene of Drosophila melanogaster codes for a variant tubulin isoform which is expressed at two distinct times during development: (1) during midembryogenesis from 8-16 hr postfertilization, and (2) during the 4 days of pupal development. We have determined the spatial pattern of beta 3-tubulin expression by localizing the beta 3 mRNA in paraffin sections using a 3' message-specific RNA probe and by localizing the beta 3 protein using a polyclonal antibody specific for Drosophila beta 3-tubulin. During embryogenesis beta 3 is restricted to and is expressed in all of the developing muscles. During pupal development beta 3 is also expressed at high levels in developing adult muscles. In addition, early in pupal development beta 3 is expressed in the imaginal discs, while at later times beta 3 is expressed in the epidermal cells of the wing blade, the optic lobe, the ovaries, and the testes. The expression of beta 3 tubulin ceases by the end of pupal development in all of these tissues except the ovaries and testes where expression persists into the adult. In both developing muscles and wings our results indicate that beta 3-tubulin is utilized in populations of specialized but transient cytoskeletal microtubules which are involved in establishing the final form of the tissue.     

Sequencing and exon mapping of the inositol 1,4,5-trisphosphate receptor cDNA from Drosophila embryos suggests the presence of differentially regulated forms of RNA and protein.

A single gene appears to code for the inositol 1,4,5-trisphosphate receptor (itpr) in Drosophila melanogaster, as compared to three known genes in mammals. Expression of the itpr gene in Drosophila occurs in a wide range of tissues and developmental stages, suggesting its requirement during diverse cellular and physiological processes. A head cDNA for the Drosophila IP3R has previously been cloned and sequenced. Here we present and analyse the sequence of cDNAs encoding the complete IP3R, obtained from embryonic stages. The embryonic cDNA is 10525bp long and is a splice variant of the head cDNA. It differs from the latter in three main respects. It has longer 5' and 3' untranslated regions, two potential casein kinase II sites are missing in the embryo form and it contains an alternate exon which results in the replacement of three residues (VHF) in the head form by five residues (GVGHSV) in the embryo form. The significance of these changes is discussed. An exon-intron map of the gene derived from sequencing of intron-containing genomic fragments is also presented. The gene has a total of 11 introns, of which more than half are clustered in a region of the modulatory domain of the IP3R.     

Inositol 1,4,5-trisphosphate receptor expression in cardiac myocytes

Calcium release from intracellular stores is the signal generated by numerous regulatory pathways including those mediated by hormones, neurotransmitters and electrical activation of muscle. Recently two forms of intracellular calcium release channels (CRCs) have been identified. One, the inositol 1,4,5-trisphosphate receptors (IP3Rs) mediate IP3-induced Ca2+ release and are believed to be present on the ER of most cell types. A second form, the ryanodine receptors (RYRs) of the sarcoplasmic reticulum, have evolved specialized functions relevant to muscle contraction and are the major CRCs found in striated muscles. Though structurally related, IP3Rs and RYRs have distinct physiologic and pharmacologic profiles. In the heart, where the dominant mechanism of intracellular calcium release during excitation-contraction coupling is Ca(2+)-induced Ca2+ release via the RYR, a role for IP3-mediated Ca2+ release has also been proposed. It has been assumed that IP3Rs are expressed in the heart as in most other tissues, however, it has not been possible to state whether cardiac IP3Rs were present in cardiac myocytes (which already express abundant amounts of RYR) or only in non- muscle cells within the heart. This lack of information regarding the expression and structure of an IP3R within cardiac myocytes has hampered the elucidation of the significance of IP3 signaling in the heart. In the present study we have used combined in situ hybridization to IP3R mRNA and immunocytochemistry to demonstrate that, in addition to the RYR, an IP3R is also expressed in rat cardiac myocytes. Immunoreactivity and RNAse protection have shown that the IP3R expressed in cardiac myocytes is structurally similar to the IP3R in brain and vascular smooth muscle. Within cardiac myocytes, IP3R mRNA levels were approximately 50-fold lower than that of the cardiac RYR mRNA. Identification of an IP3R in cardiac myocytes provides the basis for future studies designed to elucidate its functional role both as a mediator of pharmacologic and hormonal influences on the heart, and in terms of its possible interaction with the RYR during excitation- contraction coupling in the heart.     

Repeated immobilization stress reduces the gene expression of the type 1 and 2 IP3 receptors in stellate ganglia

Inositol 1,4,5-trisphosphate (IP(3)) is one of the second messengers produced by phosphoinositid hydrolysis and triggers IP(3) receptor (IP(3)R) mediated calcium release from intracellular pools. To determine whether immobilization stress affects the gene expression and protein level of IP(3)R in stellate ganglia, animals were immobilized once for 2h and/or for 7 days, 2h daily. After decapitation, stellate ganglia were extirpated and the gene expression of IP(3) receptors was evaluated. Protein levels of IP(3) receptor were measured by Western blot analysis using the antibody against IP(3) receptor. In the present work, we clearly show that type 1 and 2 IP(3) receptors, but not the type 3 IP(3) receptor, are expressed in stellate ganglia. Both types, type 1 and 2 IP(3) receptors, are not significantly affected by single 2h immobilization stress on mRNA and protein level. However, gene expression of both these types is significantly reduced by repeated immobilization stress for 7 days, 2h daily. The IP(3) receptor protein is reduced as well. Physiological relevance of our observations remains to be elucidated.     

Molecular cloning and characterization of the inositol 1,4,5-trisphosphate receptor in Drosophila melanogaster.

We isolated a cDNA encoding an inositol 1,4,5-trisphosphate receptor (InsP3R) of Drosophila melanogaster. The predicted Drosophila InsP3R (2,833 amino acids) has extensive sequence similarity to the mouse InsP3R. The polypeptide encoded by the cDNA was functionally expressed and showed characteristic InsP3-binding activity. The Drosophila InsP3R gene is located at the region 83A5-9 on the third chromosome and expresses throughout development but predominantly in the adult. Localization of the InsP3R mRNA in adult tissues suggests strong expression in the retina and antenna, indicating the involvement of the InsP3R in visual and olfactory transduction. In addition, the InsP3R mRNA is abundant in the legs and thorax, which are enriched with a muscular system. Such localization is apparently consistent with the quantitatively predominant sites for [3H]InsP3 binding in Drosophila and the fleshfly (Boettcherisca peregrina). The present study points to the likely functional importance of the InsP3/Ca2+ signaling system in Drosophila.     

Effect of two distinct stressors on gene expression of the type 1 IP3 receptors

Inositol 1,4,5-trisphosphate (IP3) is one of the second messengers, which triggers calcium release from intracellular pools via IP3 receptors. Previously we have shown that single immobilization stress increased gene expression of both, the type 1 and type 2 IP3 receptors (IP3R1 and IP3R2, respectively). In this study we evaluated whether long-term exposure to softer stressor (cold exposure to 4 degrees C) can affect the response to single immobilization stress. We examined modulation of the type 1 IP3 receptor gene expression by each stressor separately, and then in their combination. Rats were immobilized for 30 min and 120 min and were decapitated immediately or 3 h after immobilization. Cold stress was performed by exposure of animals to 4 degrees C temperature for 1, 7 and 28 days. To determine the effect of both stressors in combination, animals exposed to cold for 28 days were afterwards exposed to immobilization for 120 min and decapitated 3 h after the end of stressful stimulus. Our results verify that single immobilization increases the IP3R1 gene expression in left atria of rat heart, while cold stress elevates the level of gene expression only after the exposure to cold for 7 days. The exposure to cold for 28 days did not increase the gene expression of the type 1 IP3 receptor compared to control. Application of both stressors (28 days of cold exposure followed by 120 min of immobilization with subsequent 3 h rest) showed the tendency of increased IP3R1 gene expression compared to absolute, nonstressed control, but level of the type 1 IP3 receptor mRNA was significantly lower compared to mRNA levels of solely immobilized animals. Thus, cold exposure affects the response of the gene expression of the type 1 IP3 receptor to immobilization stress.     

Uranyl acetate modulates gene expression and protein levels of the type 2, but not type 1 inositol 1,4,5-trisphosphate receptors in mouse kidney

Nephrotoxic effect of uranium is already well documented. Nevertheless, little is known about the effect of uranium on calcium homeostasis and calcium transport systems. Calcium released from endoplasmic reticulum through special calcium release channels--inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs)--serves as a main source of cytosolic calcium signaling in the majority of cell types. To contribute to understanding mechanism of toxicity of the uranyl acetate (UA), we focused on modulation of the gene expression, protein levels and activity of IP3 receptor's intracellular calcium channels by UA in mouse kidney. We have found that UA did not affect mRNA and protein levels of the type 1 IP3Rs, but increased mRNA and also protein levels of the type 2 IP3 receptors in kidney. Nevertheless, IP3-induced calcium release was decreased by addition of UA. We assume that decreased activity of IP3 receptors due to the acute exposure to UA results in feedback, which triggers activation of IP3R2 expression. Thus, inhibition of calcium release and increased levels of the type 2 IP3 receptors might participate, at least partially, in UA-induced nephrotoxicity.     

Effect of thyroid hormones on the gene expression of calcium transport systems in rat muscles

It has been previously shown that modification of thyroid hormone levels have a profound impact on cardiac function, predominantly through a direct regulation of the sarcoplasmic reticulum protein levels. Nevertheless, little is known about the regulation of calcium transport systems in skeletal muscle due to the altered concentration of thyroid hormones. Thus, the goal of our study was to find out whether altered thyroid status could change the gene expression of the Na(+)/Ca(2+) exchanger (NCX), the inositol 1,4,5-trisphosphate (IP(3)) receptors and ryanodine receptors (RyRs) in slow and fast skeletal muscles of rats. A hyperthyroid state was maintained in rats by triiodothyronine (T(3)) administration, while methimazole was employed for inducing hypothyroidism. After a period of 2-10 months of T(3) treatment we observed a significant increase in mRNA levels of the NCX, RyRs and IP(3) receptors. This increase was more pronounced in the slow soleus than in the fast extensor digitorum longus (EDL) muscle. It is tempting to speculate that thyroid hormones also alter calcium concentration and thus influence the process of excitation-contraction coupling in the skeletal muscle.     

Adrenergic modulation of the type 1 IP3 receptors in the rat heart

Inositol 1,4,5-trisphosphate (IP3) receptors are calcium-releasing channels localized on the sarcoplasmic reticulum. IP3 receptors mediate the calcium mobilizing effect of a wide range of hormones, cytokines, and neurotransmitters and play an important role in variety of cell functions. The aim of this work was to study, how partial depletion of catecholamines affects the gene expression and protein levels of the type 1 IP3 receptors in rat heart. The type 1 IP3 receptor mRNA levels were studied in the left cardiac atrium and ventricle of rats treated with 6-hydroxydopamine (6-OHDA) in control and stressed conditions. The 6-OHDA produces anatomical and functional denervation resulting in decreased levels of noradrenaline and adrenaline. We also used corticoliberin (CRH) knockout mice, where secretion of adrenaline is significantly suppressed. Administration of 6-OHDA significantly decreases mRNA levels of the type 1 IP3 receptor in both, the left atrium and the left ventricle, while the gene expression of the sarcoplasmic reticular Ca2+-ATPase (SERCA 2) was unaffected. CRH knockout mice possess markedly lower levels of the type 1 IP3 receptor mRNA compared to wild-type mice in both, control and stressed conditions. These data point to the adrenergic modulation of the type 1 IP3 receptors in the rat hearts.     

Functional expression of the calcium release channel from skeletal muscle ryanodine receptor cDNA

Combined patch-clamp and fura-2 measurements were performed to study the calcium release properties of Chinese hamster ovary (CHO) cells transfected with the rabbit skeletal muscle ryanodine receptor cDNA carried by an expression vector. Both caffeine (1-50 mM) and ryanodine (100 microM) induced release of calcium from intracellular stores of transformed CHO cells but not from control (non-transfected) CHO cells. The calcium responses to caffeine and ryanodine closely resembled those commonly observed in skeletal muscle. Repetitive applications of caffeine produced characteristic all-or-none rises in intracellular calcium. Inositol 1,4,5-trisphosphate (IP3) neither activated the ryanodine receptor channel nor interfered with the caffeine-elicited calcium release. These results indicate that functional calcium release channels are formed by expression of the ryanodine receptor cDNA.     

From insulin synthesis to secretion: Alternative splicing of type 2 ryanodine receptor gene is essential for insulin secretion in pancreatic β cells

Increases in the intracellular Ca²⁺ concentration in pancreatic islets, resulting from the Ca²⁺ mobilization from the intracellular source through the ryanodine receptor, are essential for insulin secretion by glucose. Cyclic ADP-ribose, a potent Ca²⁺ mobilizing second messenger synthesized from NAD⁺ by CD38, regulates the opening of ryanodine receptor. A novel ryanodine receptor mRNA (the islet-type ryanodine receptor) was found to be generated from the type 2 ryanodine receptor gene by the alternative splicing of exons 4 and 75. The islet-type ryanodine receptor mRNA is expressed in a variety of tissues such as pancreatic islets, cerebrum, cerebellum, and other neuro-endocrine cells, whereas the authentic type 2 ryanodine receptor mRNA (the heart-type ryanodine receptor) was found to be generated using GG/AG splicing of intron 75 and is expressed in the heart and the blood vessel. The islet-type ryanodine receptor caused a greater increase in the Ca²⁺ release by caffeine when expressed in HEK293 cells pre-treated with cyclic ADP-ribose, suggesting that the novel ryanodine receptor is an intracellular target for the CD38-cyclic ADP-ribose signal system in mammalian cells and that the tissue-specific alternative splicing of type 2 ryanodine receptor mRNA plays an important role in the functioning of the cyclic ADP-ribose-sensitive Ca²⁺ release.     

Store depletion by caffeine/ryanodine activates capacitative Ca(2+) entry in nonexcitable A549 cells

Capacitative Ca(2+) entry is essential for refilling intracellular Ca(2+) stores and is thought to be regulated primarily by inositol 1, 4,5-trisphosphate (IP(3))-sensitive stores in nonexcitable cells. In nonexcitable A549 cells, the application of caffeine or ryanodine induces Ca(2+) release in the absence of extracellular Ca(2+) similar to that induced by thapsigargin (Tg), and Ca(2+) entry occurs upon the readdition of extracellular Ca(2+). The channels thus activated are also permeable to Mn(2+). The channels responsible for this effect appear to be activated by the depletion of caffeine/ryanodine-sensitive stores per se, as evidenced by the activation even in the absence of increased intracellular Ca(2+) concentration. Tg pretreatment abrogates the response to caffeine/ryanodine, whereas Tg application subsequent to caffeine/ryanodine treatment induces further Ca(2+) release. The response to caffeine/ryanodine is also abolished by initial ATP application, whereas ATP added subsequent to caffeine/ryanodine induces additional Ca(2+) release. RT-PCR analyses showed the expression of a type 1 ryanodine receptor, two human homologues of transient receptor potential protein (hTrp1 and hTrp6), as well as all three types of the IP(3) receptor. These results suggest that in A549 cells, (i) capacitative Ca(2+) entry can also be regulated by caffeine/ryanodine-sensitive stores, and (ii) the RyR-gated stores interact functionally with those sensitive to IP(3), probably via Ca(2+)-induced Ca(2+) release.     

Molecular cloning of cDNA encoding the Ca2+ release channel (ryanodine receptor) of rabbit cardiac muscle sarcoplasmic reticulum

We have cloned and sequenced cDNA encoding the Ca2+ release channel (ryanodine receptor) of rabbit cardiac muscle sarcoplasmic reticulum. The cDNA, 16,532 base pairs in length, encodes a protein of 4,969 amino acids with a Mr of 564,711. The deduced amino acid sequence is 66% identical with that of the skeletal muscle ryanodine receptor, but analysis of predicted secondary structures and hydropathy plots suggests that the two isoforms exhibit the same topology in both transmembrane and cytoplasmic domains. A potential ATP binding domain was identified at residues 2619-2652, a potential phosphorylation site at residue 2809, and potential calmodulin binding sites at residues 2775-2807, 2877-2898, and 2998-3016. We suggest that a modulator binding domain in the protein lies between residues 2619 and 3016. Northern blot analysis of mRNA from a variety of tissues demonstrated that the cardiac isoform is expressed in heart and brain, while the skeletal muscle isoform is expressed in both fast- and slow-twitch muscle. No ryanodine receptor mRNA was detected in extracts from smooth muscle or any other non-muscle tissue examined. The two receptors are clearly the products of separate genes, and the gene encoding the cardiac muscle ryanodine receptor was localized to chromosome 1.     

Expression of prostanoid receptors in human lower segment pregnant myometrium

Prostanoids, especially prostaglandin (PG) E(2), are important mediators of uterine relaxation and contractions during gestation and parturition. Inhibitors of PG formation as well as PG analogues are used to modulate uterine tonus. So far, only limited data are available regarding the expression of prostanoid receptors in human pregnant myometrium. In the present study, the expression of the receptors for PGE(2) (EP1, EP2, EP3, EP4), PGF(2alpha) (FP), prostacyclin (IP), and thromboxane A(2) (TP) in human pregnant myometrium was studied by RT-PCR, in situ hybridization and immunohistochemistry. Myometrial tissue was obtained from five women at term and not in labour and from two women who delivered preterm. Tissue specimens were excised from the upper edge of the transverse lower uterine segment incision. In all tissues analysed, EP1, EP2, EP3, EP4, FP, TP and IP receptor mRNA and protein was detected. mRNA expression for PGD(2) (DP) receptor was not detected in the majority of tissue specimens. EP1, EP2, EP4, IP, TP and FP receptor protein was detected on myometrial smooth muscle cells, whereas EP3 receptor protein was only expressed by stromal and endothelial cells. In situ hybridization experiments yielded similar results. The expression of the EP2 receptor mRNA was inversely related to gestational age. We suggest that the contractile effect of PGE(2) at term is probably mediated directly by the EP1 receptor expressed in myometrial smooth muscle cells and indirectly by the EP3 receptor expressed in stromal cells and a decrease in EP2 receptor expression.