Biochemical mechanisms of calcium mobilisation induced by mastoparan and chimeric hormone-mastoparan constructs.

Ca2+ efflux, Ca(2+)-ATPase, and membrane permeability measurements were used to investigate the biochemical mechanisms of Ca2+ release induced by mastoparan (MP) and the chimeric hormone-MP constructs incorporating galanin (galparan) or vasopressin antagonist (M375 and M391) moieties. Comparative studies utilised preparations of porcine cerebellar microsomes and rabbit skeletal muscle sarcoplasmic reticulum (SR). MP and chimeric peptides galparan, M375 and M391 induce Ca2+ release over a range of concentrations from 0.3-10 microM. Comparison of MP and three chimeric, N-terminal extended, constructs indicates that N-terminal extension modifies the biological properties of MP, producing changes in efficacy which are enzyme-isoform-specific. Biochemical studies indicate that the chimeric analogues and MP inhibit Ca(2+)-ATPases and directly activate the ryanodine receptor (RyR) to release Ca2+ from both heavy SR (HSR) and microsomes. The same peptides have no effect on the InsP3 receptor (InsP3R). Other actions that include modest changes in membrane permeability may also contribute to the Ca(2+)-mobilising action of MP and chimeric constructs.

Rat testicular myoid cells express vasopressin receptors: receptor structure, signal transduction, and developmental regulation.

Detection of the neurohypophysial hormones vasopressin (AVP) and oxytocin (OT) in the testis of several species has led to the proposal that these peptides may have a physiological role in the regulation of testicular function. Therefore, we investigated whether the contractile myoid cells of rat seminiferous tubules express functional receptors for AVP or OT and, thus, constitute a target for these hormones. This study used primary cultures of purified peritubular myoid cells derived from rats both before and after puberty. By several criteria, myoid cells prepared from adult rats expressed vasopressin receptors (VPRs). We detected specific and saturable [3H]AVP binding to a single population of sites with a Kd of 7.5 nM and a binding capacity of 145 fmol/mg protein. AVP stimulated the accumulation of inositol phosphates in a dose-dependent manner with an EC50 of 1.7 nM. Cloning and sequencing of the myoid cell VPR confirmed it to be the V1a subtype of VPR. VPR expression by myoid cells is under developmental control, as the receptors are present in the adult rat, but absent before puberty. In contrast, OT receptors were not expressed at any stage of development. Peritubular myoid cells are also responsive to endothelin-1 (ET-1), which potently stimulated phosphoinositidase-C. However, unlike AVP, the ET-1 responses were observed both before and after sexual maturity, suggesting different roles for AVP and ET-1 in the control of myoid cell function. Our data establish that the myoid cells of the adult rat seminiferous tubule are a target for AVP. This indicates an additional role for AVP in the regulation of testicular function and male fertility in the adult rat.

Three types of K(+) currents in murine osteocyte-like cells (MLO-Y4).

Osteocytes play an important role in signaling within bone. Communication of osteocytes with each other and with bone lining cells may have a function in mineral homeostasis and mechanotransduction. However, very little is known of the expression of ion channels in these cells. Using the whole-cell patch-clamp technique, we have detected three types of K(+) currents in the mouse osteocyte-like cell line MLO-Y4. The most commonly observed current (48% of cells) activated rapidly (20 msec) in response to depolarizing steps from -40 mV and exhibited voltage-dependent inactivation. The current was inhibited by 20 mmol/L tetraethyl ammonium (TEA) and abolished by intracellular 2 mmol/L 4-aminopyridine (4-AP). Biophysical and pharmacological characteristics of the current differed from those of inactivating K(+) currents in osteoblastic cells. In 22% of cells, a slowly activating, voltage-activated current was observed (threshold at 20-30 mV). This current was TEA insensitive, was abolished by intracellular application of 2 mmol/L 4-AP, and was strongly inhibited by apamin, a selective inhibitor of small conductance (SK) Ca(2+)-activated K(+) channels. A third current developed during whole-cell dialysis (37% of cells). This current showed little voltage sensitivity. It was abolished by intracellular application of 2 mmol/L 4-AP, high-extracellular Ba(2+) (108 mmol/L), or by inclusion of ATP in the intracellular solution, but was insensitive to TEA, apamin, Cs(+), and glibenclamide. None of these currents was affected by replacement of chloride with acetate in the bath or pipette salines. Reverse-transcription polymerase chain reaction confirmed the presence of mRNA for the types 1 and 2 SK channels, but message for the large conductance (BK) Ca(2+)-activated K(+) channel was not detected in these cells. Message for the sulphonylurea receptor SUR2, a subunit of glibenclamide-insensitive ATP-dependent K(+) channels (K(ATP)), was also detected, but the glibenclamide-sensitive SUR1 subunit was not. These data are the first descriptions of SK- and ATP-sensitive, glibenclamide-insensitive channels in cells of osteoblastic lineage. Our findings are consistent with a change in K(+) channel expression during differentiation from osteoblast to osteocyte. K(+) channels of osteocytes will contribute to maintenance of the cell membrane potential and thus may participate in mechanosensitivity and osteocyte intercellular communication. In addition, they may be involved in homeostatic maintenance of the extracellular fluid occupying the periosteocytic space.

Design and synthesis of heterofunctional V1a-selective vasopressin receptor ligands with lysine at position 9.

A peptide analogue of [8-arginine]vasopressin (AVP) with Lys substituted for Gly at position 9 ([d(CH2)5Tyr(Me)2LysNH2(9)]AVP; ALVP) has been synthesized as a precursor for the production of heterofunctional vasopressin receptor ligands. Three heterofunctional ligands have been prepared by attaching biotin and a photoreactive cross-linker capable of iodination, either alone or in combination, to the epsilon-amino group of Lys at position 9 in ALVP. The binding characteristics of these novel ligands have been determined at the V1a and V2 vasopressin receptors by employing membrane preparations of rat liver and kidney respectively. All of the analogues synthesized during the course of this study bound selectively, and with high affinity, to the V1a vasopressin receptor subtype. Our results demonstrate that the strategies described in this paper provide a convenient means of synthesizing heterofunctional vasopressin receptor ligands with preservation of subtype-specific, high affinity binding characteristics. These parameters establish the potential value of the analogues as probes for investigating V1a receptor structure and function.

Novel strategies for the design of receptor-selective vasopressin analogues: Aib-substitution and retro-inverso transformation.

1. We determined the pharmacological profile of novel backbone-modified peptides designed as protease-resistant, selective analogues of AVP. Binding affinities of peptides were determined at both V1A and V2 subtypes of vasopressin receptor (VPR). Biological potencies of selected peptides were tested in pressor and antidiuretic bioassays. 2. Substitution of the achiral alpha-aminoisobutyric acid (Aib) at position 4 or 7 of AVP produced peptides that selectively bound the V2 VPR. Both [Aib4]AVP (140 IU mg-1) and [Aib7]AVP (36 IU mg-1) are selective antidiuretic agonists with little or no activity in uterotonic and pressor assays. 3. [Aib4] and [Aib7] derivatives of the linear V1A-selective antagonist [PhaaDTyr(Et)2Arg6Tyr(NH2)9]AVP bound selectively and with high affinity (Kd 0.51 and 4.1 nM respectively) to the V1A VPR. Bioassays confirmed that these peptides were potent antivasopressor agents (pA2 8.10 and 8.36 respectively). 4. A total retro-inverso strategy was used to prepare protease-resistant mimetics of both AVP and linear V1A-selective antagonists. Cyclic retro-inverso mimetics of AVP did not bind either V1A or V2 VPRs. In contrast, rationally designed retro-inverso mimetics of linear V1A-selective antagonists selectively bound the V1A VPR. 5. Our findings indicate novel methods to improve the pharmacodynamic and pharmacokinetic parameters of neurohypophysial hormone analogues which could be equally applicable to other peptide-receptor systems.

Pharmacological characterization of linear analogues of vasopressin generated by the systematic substitution of positions 1 and 6 by L-amino acids.

Eighteen linear analogues of [Arg8]vasopressin (AVP) were synthesized by systematically substituting the cysteine residues at positions 1 and 6 with a range of L-amino acids. Screening by competition ligand binding revealed that the combinations of amino acid residues tolerated at these positions was very restricted with respect to retention of vasopressin receptor (VPR) binding. Consequently, only three of the eighteen analogues investigated, [Pro1,Met6]AVP, [Gly1,Met6]AVP and [Phe1,Lys6]AVP, bound to the V1a receptor. Furthermore, these three peptides were all selective for the V1a receptor rather than the V1b, V2 and vasotocin receptors. In addition, although very homologous to the natural agonist, these analogues were in fact antagonists at V1a receptors. These data provide insights into the biophysical requirements at positions 1 and 6 of linear ligands for binding to V1a receptors and furthermore, supply clues to the nature of the receptor:ligand interaction.

A selective biotinylated probe for V1a vasopressin receptors.

We have designed and synthesized a biotinylated vasopressin antagonist which is a selective probe for studying the V1a subtype of vasopressin receptor. Initially we synthesized the novel vasopressin analogue d(CH2)5Tyr(Me)2LysNH2(9)AVP (ALVP). Biotinamidocaproate was subsequently coupled to the epsilon-amino group of ALVP to generate the novel biotinylated probe d(CH2)5Tyr(Me)2Lys(N epsilon-biotinamido-caproate)NH2(9)AVP (ALBtnVP). Pharmacological characterization of ALVP and ALBtnVP established that both ligands were high affinity antagonists at V1a receptors, and that both displayed marked V1a/V2 selectivity. The observation that receptor-bound ALBtnVP was bi-functional, and thereby able to bind conjugated derivatives of avidin or streptavidin, allowed ALBtnVP to be utilized as a selective probe for V1a receptors. This strategy allowed the visualization of V1a receptors on the surface of WRK-1 cells and hippocampal neurons, by using streptavidin-gold with electron microscopy and fluorescein-avidin with light microscopy. We conclude that ALBtnVP is a useful probe for V1a receptors.

Characterization of G protein-coupled receptors expressed by ECV304 human endothelial cells.

The aims of this study were to characterize G protein-coupled receptors endogenously expressed by ECV304 human endothelial cells, and to determine the utility of this transformed cell line as a vehicle for the expression of cloned receptors. Cellular responses to a broad range of agonists were determined by measuring changes in the intracellular content of second messengers (inositol phosphates and cyclic adenosine monophosphate). These studies identified H1 histamine receptors, P2U-purinoceptors and lysophosphatidic acid receptors which are functionally coupled to phosphoinositidase C. G protein-coupled receptors which bind adenosine (A2 receptor), calcitonin, and adrenaline (beta-adrenoceptor), and markedly stimulate adenylyl cyclase, are also endogenously expressed by ECV304. Agonists which did not stimulate ECV304 cells are: angiotensin II, angiotensin1-7, bombesin, bradykinin, desArg9-bradykinin, carbachol, endothelin-1, neurotensin, serotonin, substance K, substance P, thrombin and vasopressin. The rat Via vasopressin receptor was expressed by lipofection in two antibiotic-resistant clonal lines and expression confirmed by measuring agonist-induced changes in inostol phosphate production. We conclude that the ECV304 cell line is a suitable in vitro system to study the signal transduction pathways of some endogenous G protein-coupled receptors known to modulate endothelial function in vivo. ECV304 is also appropriate for the expression and functional characterization of cloned receptor proteins.

Fluorescent and biotinylated probes for B2 bradykinin receptors: agonists and antagonists.

Peptide ligands carrying additional reporter groups are valuable research tools to facilitate biochemical and pharmacological studies of G protein-coupled receptors. B2 bradykinin receptors, widely distributed in mammalian tissues, regulate many physiological systems and are therapeutic targets. Acylation of the amino-terminus of bradykinin (BK) and a B2a-selective antagonist produced ligands derivatized with biotinamidocaproate or 7-Amino-4-methylcoumarin-3-acetate. These fluorescent and biotinylated peptides bound with high affinity to bovine and rodent B2 receptors. Analysis of second messenger production confirmed that fluorescent and biotinylated analogs of BK were B2 receptor agonists whereas derivatives of DArg0[Hyp3,DPhe7,Leu8]BK were BK receptor antagonists. The complimentary properties of these selective receptor probes will be useful in studying B2 receptor localization, expression and desensitization.

Effects of vasopressin-mastoparan chimeric peptides on insulin release and G-protein activity.

Two chimeric peptides, consisting of the linear vasopressin receptor V1 antagonist PhAc-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr, in the N-terminus and mastoparan in the C-terminus connected directly (M375) or via 6-aminohexanoic acid (M391), have been synthesised. At 10 microM concentration, these novel peptides increased insulin secretion from isolated rat pancreatic islet cells 18-26-fold at 3.3 mM glucose and 3.5-5-fold at 16.7 mM glucose. PTX pretreatment of the islets decreased the peptide-induced insulin release. M375 and M391 bind to V1a vasopressin receptors with affinities lower than the unmodified vasopressin antagonist, but with K(D) values of 3.76 nM and 9.02 nM, respectively, both chimeras are high affinity ligands. The GTPase activity and GTPgammaS binding in the presence of these peptides has been characterised in Rin m5F cells. Comparison of the influence of the peptides M375 and M391 on GTPase activity in native and pertussis toxin-treated cells suggests a selective activation of G alpha(i)/G alpha(o) subunits, combined with a suppression of other GTPases, primarily G alpha(s). However, the GTPgammaS binding data show that the peptides retain some of the activating property even in PTX-treated cell membranes. In conclusion, the conjugation of mastoparan with the V1a receptor antagonists produce peptides with properties different from the parent peptides that could be used to elucidate the role of different G proteins in insulin release.

Probing the V1a vasopressin receptor binding site with pyroglutamate-substituted linear antagonists.

We have synthesized eight analogues of the linear vasopressin antagonist DTyr(Et)2-Phe3-Gln4-Asn5-Arg6-Pro7-Arg8-Tyr(NH2)9 substituted with L-, or D-, pyroglutamate at position-1, Asn or Val at position-4 and Arg or Met at position 6. All of these peptides bound to the V1a vasopressin receptor with affinities ranging 33.6-5, 470 nM. Of this series, only two peptides, [LpGlu1Val4Arg6Tyr(NH2)9]AVP Kd = 48.4 nM and [DpGlu1Val4Arg6Tyr(NH2)9]AVP Kd = 691 nM, bound to the V2 vasopressin receptor. All of the neurohypophysial hormone receptors studied (V1a VPR, V2 VPR and OTR) were found to be stereoselective with respect to the N-terminal pGlu residue. The effect on binding characteristics of L-pGlu1 and D-pGlu1 analogues was dependent on both the sequence of the peptide and on the receptor subtype in question. From these data we found that peptide 5, which has the structure DpGlu-DTyr(Et)-Phe-Val-Asn-Arg-Pro-ARg-Tyr(NH2), exhibited the highest V1a/OTR selectivity reported to date (V1aVPR Kd = 82 nM; OTR no binding at 10 microM). As such, peptide 5 will provide useful leads to the development of ligands with enhanced V1a/OTR selectivity. The binding affinity and hydrophobicity of pyroglutamate-substituted peptides was compared with previously characterized V1a receptor antagonists which contained a range of position-1 substitutions. The hydrophobicity of both cyclic and linear antagonists was markedly increased relative to the agonists AVP and [Phe2Orn8]VT but increased hydrophobicity alone did not exclusively lead to high affinity antagonists. Data presented support the contention that in addition to a general increase in hydrophobicity/lipophilicity, position-1 influences the pharmacophore of vasopressin antagonists by providing molecular determinants for ligand/receptor interaction.

The many futures for cell-penetrating peptides: how soon is now?

Studies of CPPs (cell-penetrating peptides), sequences that are also commonly designated as protein transduction domains, now extend to a second decade of exciting and far-reaching discoveries. CPPs are proven vehicles for the intracellular delivery of macromolecules that include oligonucleotides, peptides and proteins, low-molecular-mass drugs, nanoparticles and liposomes. The biochemical properties of different classes of CPP, including various sequences derived from the HIV-1 Tat (transactivator of transcription) [e.g. Tat-(48-60), GRKKRRQRRRPPQ], and the homeodomain of the Drosophila homeoprotein Antennapaedia (residues 43-58, commonly named penetratin, RQIKIWFQNRRMKWKK), also provide novel insights into the fundamental mechanisms of translocation across biological membranes. Thus the efficacy of CPP-mediated cargo delivery continues to provide valuable tools for biomedical research and, as witnessed in 2007, candidate and emerging therapeutics. Thus it is anticipated that the further refinement of CPP technologies will provide drug-delivery vectors, cellular imaging tools, nanoparticulate devices and molecular therapeutics that will have a positive impact on the healthcare arena. The intention of this article is to provide both a succinct overview of current developments and applications of CPP technologies, and to illustrate key developments that the concerted efforts of the many researchers contributing to the Biochemical Society's Focused Meeting in Telford predict for the future. The accompanying papers in this issue of Biochemical Society Transactions provide additional details and appropriate references. Hopefully, the important and eagerly anticipated biomedical and clinical developments within the CPP field will occur sooner rather than later.

Hepatic vasopressin receptors (VPRs) exhibit species heterogeneity--absence of VPRs in sheep liver.

1. We have studied the binding characteristics of the hepatic VPRs expressed by rat, cow, pig, sheep and human and have demonstrated species heterogeneity. 2. These species differences are manifested as variation in the VPR capacity (rat > cow > pig > human > sheep), and in the affinity of these receptors for their natural ligand AVP (rat = human > pig = cow). 3. A single class of VPRs, with high affinity for AVP, is present in rat, cow, human and pig liver. In contrast, ovine hepatocytes do not express a VPR. 4. The affinity of the V1a-selective antagonist d(CH2)5Tyr(Me)2AVP is highly dependent on the species utilised, such that rat > human > cow.

Molecular pharmacology of V1a vasopressin receptors.

1. Vasopressin, a mammalian neurohypophysial peptide hormone, has diverse physiological actions. 2. Pharmacological studies, using a range of mammalian tissues, have identified three subtypes of vasopressin receptor. 3. The V1a subtype of vasopressin receptor is widely distributed and mediates many central and peripheral actions of vasopressin. 4. The development of subtype-selective vasopressin analogues has provided valuable tools for pharmacological and physical studies of the V1a receptor protein. 5. Pharmacological differences indicate species heterogeneity in the characteristics of V1a receptors and in the expression of hepatic V1a receptors. 6. The cloning of neurohypophysial hormone receptor proteins allows structural and functional comparison of the V1a vasopressin receptors with other G-protein-coupled receptors.

Molecular recognition of peptide and non-peptide ligands by the extracellular domains of neurohypophysial hormone receptors.

This study was designed to ascertain whether the extracellular loops of vasopressin/oxytocin receptors bind ligands and, if so, to locate the molecular determinants of this ligand-receptor interaction. Ligand-binding studies were employed using a rat liver V1a vasopressin receptor preparation and both peptide and non-peptide receptor ligands. Synthetic peptides corresponding to defined regions of the extracellular surface of the neurohypophysial hormone receptors recognized radioligands. These receptor mimetics inhibited the binding of radioligands to the V1a receptor with apparent affinities (pKi) ranging from 3.1 to 6.75. The same mimetics had no effects on the binding of angiotensin II to the rat AT1 receptor, indicating specificity for V1a receptor ligands. A mimetic peptide (DITYRFRGPDWL) of the first extracellular loop (ECII) of the V1a vasopressin receptor also inhibited vasopressin-stimulated, but not angiotensin II-stimulated, glycogen phosphorylase in isolated rat hepatocytes. In contrast, scrambled ECII mimetics displayed greatly reduced affinity for vasopressin. In addition, the role of peptide side-chain versus main-chain atoms in the binding of ligands by vasopressin receptors was addressed using retro-inverso peptide mimetics. Our findings indicate a precise orientation of the extracellular receptor surface (particularly the ECII domain) which facilitates the initial 'capture' of both peptide and non-peptide ligands. Moreover, the data indicate that the main-chain atoms of both a major binding-site determinant in the first extracellular loop of the receptor and the neurohypophysial hormones contribute significantly to the ligand-receptor interaction. These findings also suggest that soluble receptor-binding domains have therapeutic potential.

Bay K 8644 induced necrosis in murine skeletal muscle in vitro: myofibre breakdown precedes significant alterations of intracellular [Ca] or pH.

The effect of the Ca2+-channel agonist Bay K 8644 (1 mumol/l) on the ultrastructure, Ca2+-homeostasis, pH and membrane potential of murine diaphragm muscle, in vitro, has been investigated. Treatment with Bay K 8644 in a standard physiological saline, for 1-2 h, induced swelling of the muscle mitochondria and minor damage to the myofibrils. Ultrastructural Ca-localisation by antimonate precipitation revealed no differences between treated and control preparations. Accompanying the structural changes there was a small, non-significant increase in muscle Ca content. In EGTA-buffered (Ca-free) standard saline the induction of damage was not inhibited. When [K+]o was raised to 20 mmol/l, a procedure that approximately halved the resting potential, Bay K 8644 induced severe ultrastructural damage within 1 h, and complete cellular necrosis within 2 h. Induction of myopathy was unaffected by synaptic blockade (150 mumol/l D-tubocurarine). Necrosis was accompanied by depolarisation of membrane potential (Em) and increased antimonate precipitation in the sarcoplasm, and was abolished by buffering of [Ca2+]o with EGTA. However, muscles did not develop tension and measurements of both total Ca and [Ca2+]i suggest that cellular Ca2+ buffering was not seriously impaired until 2 h after Bay K 8644 application. Measurement of sarcoplasmic pH revealed no significant change during fibre necrosis. It is proposed that in partially depolarised preparations Bay K 8644 acts on a Ca2+-channels in the cell membrane, probably the T-tubules, to induce muscle necrosis through enhanced influx of Ca2+. However, muscle necrosis occurs before significant elevation of [Ca2+]i and does not require sarcoplasmic acidification.

Intestinal mast cell tumor in a cat: presentation as eosinophilic enteritis.

A 10-year-old, neutered male domestic shorthair was diagnosed as having eosinophilic enteritis following two separate surgical biopsies. Although treatment was aggressive and a partial response was seen, the cat's condition deteriorated. Postmortem histopathology revealed an intestinal mast cell tumor.