b1 and b2 bradykinin receptors encoded by distinct mrnas

Journal of Neurochemistry Raven Press, Ltd., New York 0 1994 International Society for Neurochemistry

B, and B, Bradykinin Receptors Encoded by Distinct mRNAs

Michael Webb, Peter McIntyre, and Elsa Phillips

Sundoz Institute for Medical Research, London, England

Abstract: Bradykinin receptors have been subdivided into at least two major pharmacological subtypes, 6, and 6,. The cDNAs encoding functional 6, receptors have recently been cloned, but no molecular information exists at present on the 6, receptor. In this article, we describe experiments examining the possible relationship between the mRNAs encoding the 6, and B, types of receptor. We showed previously that the human fibroblast cell line W138 expresses both B, and B, receptors. In this report, we describe oocyte expression experiments showing that the B, receptor in W138 human fibroblast cells is encoded by a distinct mRNA -2 kb shorter than that encoding the B, receptor. We have used an antisense approach in conjunction with the oocyte expression system to demon- strate that the two messages differ in sequence at several locations throughout the length of the B, sequence. Taken together with the mixed pharmacology exhibited in some expression systems by the cloned mouse receptor, the data indicate that B,-type pharmacology may arise from two independent molecular mechanisms. Key Words: Bradykinin receptors-Oocyte expression-Antisense oligonucleotide-mRNA analysis. J. Neurochem. 62,1247-1 253 (1 994).

Bradykinin (BK; Arg-Pro-Pro-Gly-Phe-Ser-Pro- Phe-Arg) is a member of a small family of peptide hormones, the kinins. Both BK and the related peptide kallidin (Lys-BK) are released from high-molecular-weight precursors, the kininogens, which are syn- thesized in the liver and released into the circulation. Activation of proteases at sites of tissue damage results in the cleavage of the kininogen and the release of the peptides (reviewed by Erdos, 1979). The peptides have various effects, including vasodilation, increased vascular permeability, contraction of smooth muscle, and direct stimulation of primary afferent sensory neurones (Baccaglini and Hogan, 1983; Steranka et al., 1988). In addition, kinins have been shown to be mitogenic for some fibroblast cells (Owen and Vil- lereal, 1983). These effects have suggested that the peptides are involved in orchestrating the response to tissue injury, and kinins have also been implicated in the pathophysiology of inflammation.

Regoli et al. (1977) and Regoli and Barabe (1980) proposed a subdivision of the kinin receptors into two classes. B, receptors have a high affinity for BK and

kallidin but a very low affinity for either of these kinins when the carboxy-terminal arginine is removed (des-Arg9-BK and des-Arg’O-kallidin). B, receptors, in contrast, have a higher affinity for these truncated peptides than for the intact sequence. There are several selective antagonists of B, receptors, including ~-phe’-BK, NPC 567, and the recently described highly potent peptide HOE 140 (Hock et al., 1991). B, responses can be specifically antagonised by Leu8, des-Arg9-BK (reviewed by Farmer and Burch, 1992).

B, receptors are quite widely distributed in many normal tissues. B, receptors have a more limited dis- tribution and are found primarily on the vasculature. It has been shown that in pathological situations, the expression of B, receptors may be induced or increased in various tissues, including blood vessels (Marceau et al., 1984), intestinal tissue (Boschcov et al., 1984), and bladder (Marceau et al., 1980). Inhibi- tors of protein synthesis have been shown to block this expression (Regoli et al., 1978; Whalley et al., 1983). This indicates that protein synthesis is required for the appearance of the new receptor pharmacology, although there are no reliable binding data to prove that the increased responsiveness to the B,-selective agonist des-Arg9-BK is correlated with an increased number of specific binding sites. The inducibility of B, receptors after injury suggests that they may have a specific role in inflammatory situations and chronic pain states.

McEachern et al. (199 1) cloned a B, BK receptor- encoding cDNA. Northern blots of RNA derived from various tissues expressing BK B, receptors yielded several bands when probed with the cloned B, receptor cDNA. However, Southern blots of genomic DNA from rat, human, and guinea pig sources yielded data consistent with the presence of only a single gene with high homology to the cloned sequence. The multiple RNA species revealed by northern blotting suggest that alternative splice variants

Received July 5, 1993; revised manuscript received August 16, 1993; accepted August 16, 1993.

Address correspondence and reprint requests to Dr. M. Webb at Sandoz Institute for Medical Research, 5 Gower Place, London WCl E 6BN, U.K.

Abbreviations used: BK, bradykinin; SHT, serotonin.

1247

1248 M. WEBB ET AL.

may be derived from the same gene or that different poly(A) addition signals are used. However, the Southern blot data indicate that if the B, receptor is encoded by a distinct gene, it cannot be highly homologous to the B,-encoding gene.

We showed previously (Phillips et al., 1992) that injection of Xenopus oocytes with poly(A)+ RNA derived from the human fibroblast cell line W138 re- sulted in the expression of both B, and B, receptors by the oocytes. We have previously cloned, sequenced, and characterised the B, BK receptor from WI38 cells (McIntyre et al., 1993). We wished to investigate the physical relationship between the B, and B, receptor- encoding RNAs. In this article, we describe experiments in which we have used the Xenopus expression system to show that the mRNA encoding the B, receptor in these cells is -2 kb shorter than that encoding the B, receptor. In addition, we have used antisense oligonucleotides based on the human B, receptor sequence to show that the two mRNAs differ in sequence at seven sites throughout the coding region. These observations show that the mRNA encoding B, receptors in these cells is distinct, both in size and in coding sequence, from that encoding B, receptors.

MATERIALS AND METHODS

Cell culture Nontransformed WI38 cells were obtained from Flow

Laboratories. They were maintained in Dulbecco’s modi- fied Eagle’s medium supplemented with 10% foetal calf serum and passaged by 1 :2 serial subculture.

RNA preparation Total RNA was prepared by the guanidinium isothio-

cyanate method of Chomczynski and Sacchi (1987). Poly(A)+ RNA was prepared by one or two cycles of binding and elution from oligo-dT cellulose columns (Sambrook et al., 1989). Purified poly(A)+ RNA was precipitated with ethanol and dissolved in water. Nucleic acid concentrations were estimated by absorbance at 260 nm. Sucrose density gradient fractionation of poly(A)+ RNA was camed out as described previously (Phillips et al., 1992).

Oligonucleotide synthesis and in vitro transcription Oligonucleotides with conventional phosphodiester bonds

were synthesised on an Applied Biosystems PCRmate ma- chine. After deprotection, the oligonucleotides were purified by use of an OPC cartridge (Applied Biosystems). The purified oligonucleotides were treated with proteinase K (100 pg/ml) at 55°C for 60 min to remove contaminating RNase. They were extracted twice with an equal volume of 1 : 1 phenol/chloroform and twice with an equal volume of chloroform. After reprecipitation, with ethanol, they were dissolved in water, and the concentration was adjusted to 2 mg/ml.

The sequences of oligonucleotides used in this study were as follows: AS2 (initiation codon and succeeding 25 bases of rat B, receptor), CCA GGG CTT GCG TGG TGA TGT TGA ACA T; AS3 (initiation codon and succeeding 24 bases of rat serotonin 5-HT1, receptor), CAG GAG CGA GCG CAC CGC GTT GCC AAG; and H2-H9, located at various positions on the human B, receptor sequence, as

indicated in Fig. 3-H2, GGT CCC GTT AAG AGT GGG CC; H3, GGT GTT GAG CCA GCC CAG CC; H4, GCA GGC CAG GAT CAG GTC TG; H5, CTT CAT GGT CCG GAA CAC CAG, H6, CTG ATG ACA CAA GCG GTG AC; H7, GCT GAT CTG GAA GGG CAG CC; and H9, GGC GAT CTG TGT GAT TAC ATC G.

In some experiments cRNAs transcribed from a cloned rat 5-HT,, receptor cDNA (Julius et al., 1988) were used. Transcription from 1 pg of linearised DNA was camed out using a Stratagene transcription/capping kit according to the manufacturer’s instructions. After DNase treatment and extraction with phenol/chloroform and chloroform, the cRNA was precipitated with ethanol and dissolved in 5 p1 of water. Dilutions of transcript quoted in the text refer to this initial solution.

Oocyte injections and electrophysiology Xenopus oocytes were obtained and studied as described

(Phillips et al., 1992). Oocytes were injected with 50 nl of RNA or RNA/oligonucleotide mixtures by means of fine glass syringes with tip diameters of - 15-30 pm.

Antisense experiments were performed by mixing appropriate oligonucleotides and RNA in vitro and then injecting the mixture into the oocytes. In some experiments the poly(A)+ RNA from the WI38 cells was mixed with a diluted transcript of a 5-HT1, cDNA. This served as a control on the specificity of inhibition of expression by the oligonucleotides and also as a positive control that oocytes had been properly injected. A pool of poly(A)+ RNA encoding both B,- and B,-type responses was selected from the sucrose gradient for these experiments. The final concentrations of the components of these mixtures were as follows: WI38 mRNA, 0.75 or 1 &pl; oligonucleotide, 1 pg/pl; and 5-HT transcript, 1: 1,000. In some experiments, an oligonucleotide specific for the 5-HT,, receptor was used as a control. In other experiments, a control mixture consisted of appropriately diluted W138 poly(A)+ RNA and 5-HT transcript, with no added antisense oligonucleotide.

Northern blotting The sodium phosphate transfer method recommended

by NEN for use in conjunction with GeneScreen mem- branes was used. Poly(A)+ RNA samples (6 pg) were resolved on formaldehyde gels and capillary-blotted onto the nylon membrane. After prehybridisation, the blot was hy- bridised overnight with a 1.3-kb probe of full-length rat B, receptor-encoding DNA ( lo6 cpm/ml). The blot was washed at 65°C in 2 X SSC for 10 min and in 0.2 X SSC at room temperature for 15 min ( 1 X SSC is 0.15 M NaCl and 0.0 15 M sodium citrate, pH 7.0) and exposed to x-ray film.

RESULTS

Size fractionation of B,- and B,-encoding poly(A)+ RNAs

We fractionated WI38 mRNA on sucrose gradients in several independent experiments. The fractions were injected individually into oocytes, and these were assayed for their responsiveness to BK and to des-Arg9-BK. Oocytes were injected with 50 nl of the RNA fractions. Three days later they were voltage- clamped at -55 mV and exposed first to des-Arg9-BK (2 pM), followed by BK (100 nM).

Figure 1 shows the responses obtained from oocytes

J. Neurochem., Vol. 62. No. 4. 1994

1249 B, AND B2 BK RECEPTORS ENCODED BY DISTINCT rnRNAS

des Arg9 Bk Bk

13

des ArgY Bk Bk

14

des ArgY Bk Bk I I

des ArgY Bk - B ?

des Argg Bk

;" FIG. 1. Recordings obtained from oocytes injected with sucrose gradient-fractionated poly(A)+ RNA fractions. Fraction 13 con- tains RNA species of -4.5 kb, and the size of the RNA decreases from fraction 13 to fraction 17. The oocytes were voltage- clamped at -55 mV, and the membrane currents evoked in response to des-Arge-BK (2 f l and BK (100 nM) were recorded.

injected with fractions from a sucrose gradient. 00- cytes injected with RNA fractions above -4 kb were able to respond to BK with an inward current, although these oocytes were essentially unresponsive to the B,-selective agonist des-Arg9-BK. This is in agree- ment with our own previous estimate of the size of the B,-encoding RNA species from rat uterus and NGlO8-15 cells (Phillips et al., 1992). It also agrees with the observations of McEachern et al. (1991), whose B, receptor-encoding cDNA appears to be a full-length clone of a 4-kb mRNA species.

Oocytes injected with mRNA from the middle region of the gradient (fractions 14 and 15 in Fig. 1) were responsive to both agonists. However, oocytes injected with fractions from the lower end of the gra-

dient were able to give robust responses to des-Arg9- BK but were essentially unresponsive to BK (Fig. 1, fractions 16 and 17). The gradients therefore effected a clear separation between the mRNA species conferring responsiveness to BK and to des-Arg9-BK. The size of the RNA encoding the B, receptor is known from previous experiments to be -4 kb. Using resid- ual rRNA in the fractions as markers, we estimate that the mRNA species conferring responsiveness to des-Arg9-BK is -2 kb in length.

Specificity of antisense oligonucleotide inhibition of receptor expression

We injected oocytes with a mixture of RNAs encoding both BK receptors and 5-HT,, receptors. The RNA encoding the BK receptor was either poly(A)+ RNA from NG108-15 cells (Phillipset al., 1992) or in vitro transcripts of a cloned rat B, receptor cDNA (McIntyre et al., 1993). These RNAs were mixed with in vitro transcript from a rat 5-HT,, receptor cDNA. Antisense oligonucleotides based on the initiation codon and succeeding 24 or 25 nucleotides of either the 5-HT,, receptor or the rat B, receptor, respectively, were added, and the mixtures were injected into oocytes. These oocytes were challenged with both BK (100 nM) and with 5-HT ( I00 nM). Examples of the responses obtained are shown in Fig. 2.

The 5-HT receptor antisense oligonucleotide completely inhibited the response to 5-HT while sparing the response to BK (n = 3; BK responses were 100- 500 nA). Similarly, when cRNA transcript was used to generate the BK response, the BK B2 receptor antisense oligonucleotide completely eliminated the response to BK, whereas responses to 5-HT remained intact (n = 4; 5-HT responses were between 100 and 1,000 nA).

In the second series of experiments, poly(A)+ RNA from NG108-15 cells was used to generate the BK response, and the anti-BK B, receptor oligonucleotide was used. 5-HT signals were observed in all of these oocytes (n = 8; 5-HT responses were between 400 and 2,000 nA). Six to eight oocytes in this series gave no response to BK. Of the two remaining oocytes, one gave a 25 nA response to BK, whereas the other gave slight oscillations on application of BK.

Inhibition of B, but not B, receptor expression by antisense oligonucleotides

We wished to determine whether inhibition of B, receptor expression by antisense oligonucleotides would invariably be accompanied by a failure of B, receptor expression. This would indicate that the mRNAs generating both receptors are identical at the point in the sequence recognised by the antisense oligonucleotide. Alternatively, inhibition of the B, response but not the B, response would indicate that the two mRNAs are not similar enough in sequence to be recognised by the same oligonucleotide at that point in their sequences.

J Neurochem., Vol. 62, No. 4, 1994

I250 M. WEBB ET AL.

L ' I mm

A BK SHT BK

B I

BK 5HT

c 1 I

BK snr

FIG. 2. Specificity of antisense oligonucleotide inhibition of receptor expression. Oocytes were injected with RNA mixed with antisense oligonucleotide as described in Materials and Methods. A. Oocyte injected with cRNA transcripts of the rat B, BK receptor and the rat 5-HT1, receptor cDNAs mixed with an antisense oligonucleotide specific for the 5-HT1, receptor. The oocyte was challenged with BK (100 nM) and 5-HT (100 nM). A second BK response could be evoked from the oocyte, which failed to respond to 5-HT. B: An oocyte was injected with the same transcripts as in A but mixed with an antisense oligonucleotide specific for the rat B, BK receptor. It was challenged with BK and 5-HT as in A. C: An oocyte was injected with a mixture of poly(A)+ RNA from NGI 08-1 5 cells and cRNA transcribed from the 5-HT,, cDNA and challenged as before. Note scale difference between B and C.

We designed a series of antisense oligonucleotides based on the human B2 receptor. The locations of these oligonucleotides along the amino acid sequence encoded by the B, nucleotide sequence are shown in Fig. 3. We used appropriately pooled sucrose gradient fractions to generate robust responses to both B,- and B,-selective agonists. Samples of pooled RNA were mixed with antisense oligonucleotides and injected into oocytes. After culture for 3 days, the oocytes were voltage-clamped and challenged with both BK ( 100 nM) and des-Arg9-BK (2 pM).

Figure 4A shows the relative levels of signal recorded in response to BK and to des-Arg9-BK from oocytes injected with pooled RNA in the absence of antisense oligonucleotide. When 1 1 control oocytes taken from different experiments were compared, the range of responses observed was 40-450 nA with BK and 5-400 nA with des-Arg9-BK, and the ratio (response to des-Arg'-BK/response to BK) vaned between 0.1 and 0.88. The variation in the relative mag- nitude of the responses was probably due to differ- ences in the precise composition of the pooled RNA

used to generate the response in different experiments.

Representative traces for three of the antisense oligonucleotides, H3, H4, and H6, are shown in Fig. 4. All three oligonucleotides abolished the ability of the oocytes to respond to BK, indicating the successful targeting of the B,-encoding mRNA by the oligonucleotides. However, in each case, the ability of the oocyte to respond to des-Arg9-BK was not inhibited. All of the oligonucleotides indicated in Fig. 3 were observed in at least three independent oocytes to behave in the same way, completely abolishing the ability of the oocytes to respond to BK, without affecting their responses to des-Arg9-BK.

Figure 4D shows the response of an oocyte injected with antisense oligonucleotide H6. This oocyte was unable to respond to BK, and it was also unresponsive to des-Arg9-BK in the presence of the B,-selective antagonist Leu', des-Arg9-BK. The oocyte was able to respond to the B,-selective agonist after washout of the antagonist. The responses to des-Arg9-BK that we have observed in oocytes treated with antisense oligonucleotides are therefore B, , as judged by the action of subtype-selective agonists and antagonists.

RNA species detected by northern blotting We examined the RNA species expressed by W138

cells by blotting mRNA and probing the blots with a rat B, receptor-encoding cDNA probe. We included samples of other tissues as controls. Rat uterus, the hybrid NG 108- 1 5 cell line, rat lung, and the W138 cell line all expressed three major bands of 6.6, 5.8, and 4 kb (Fig. 5). Bands of similar size were described in the uterus and lung by McEachern et al. (1991). W138 cells, which express both B, and B, receptors, did not express additional bands on these blots.

MLNVTLQCPTLNGTFAQSKCPQVEWLG W L N T I a

PFLWVLFVLATLENIFVLSWCLHKSSCTVAEU

LAAA D L I L A C G L P F W A I T I S E T L C R ~

NAIISMNLYSSICFLMLVSIDRYLALVKTMSMGRM

RGVRWAKLYSLVWGCTLLLSSPWVFRTMKEYS

DEGHNVTACVISYPSLIWEWTNMLLNVVGFLLPL

SVITFCTMOIMOVLRNNEMQKFKEIQTERRA~

LVVLLLFIIC WLPFOISTFLDTLHRLGILSSCQDERIl

PVITOIASFMAYSNSCLNPLVYVIVGKRFRKKSWE

WQGVCQKGGCRSEPIQMENSMGTLRTSISWRQI

HKLQDWAGSRQ

H2 H3

H4

H5

H6

H7

H9

FIG. 3. The amino acid sequence of the human 8, receptor in single letter code. Putative transmembrane domains are under- lined. The locations of the amino acids encoded by the DNA sequences used for the design of the antisense oligonucleotides are shown in italics and bold.

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B, AND B2 BK RECEPTORS ENCODED BY DISTINCT mRNAS 1251

FIG. 4. Antisense oligonucleotide inhibition of 6, but not 6, receptor expression in oocytes injected with W138 poly(A)' RNA. All oocytes were injected with W138 poly(A)+ RNA plus (A) water (control), (B) antisense oligonucleotide H3, (C) antisense oligonucleotide H4, and (D) antisense oligonucleotide H6. The control oocyte responded to BK (1 00 nnn) and des-Args-BK (2 pM), and oocytes injected with H3 and H4 responded to des-Args-BK but not to BK. The oocyte injected with H6 did not respond to BK. It failed to respond to des-Args-BK in the presence of the 8,-selective antagonist Leu', des-Argg-BK, but the same oocyte was able to respond to des- Arg8-BK when the antagonist was washed out.

I I I

Bk off des Arg9 Bk

I I I I Bk des Arg9 Bk Bk des Arg9 Bk

DISCUSSION

The Southern blotting experiments of McEachern et al. (1991) suggested that there was no gene highly homologous to that encoding the rat B, receptor in the nuclear DNA of the human, rat, or guinea pig. The experiments we describe here were undertaken to determine if mRNA encoding the B, receptor in the W138 cell line had any sequence homology with that encoding the B, receptor in the same cell line.

In this study, we have provided evidence that the two receptors are encoded by mRNAs of different sizes. The mRNA encoding the rat B, receptor is -4

A B C D

I I / / I I / I I Bk Leu8 des- "Leu8 des Arg9 Bk

Arg9 Bk w%h des Arg9 Bk

+ des Arg9 Bk

FIG. 5. Northern blot of poly(A)+ RNA prepared from NG108-15 (lane A), rat uterus (lane B), rat lung (lane C), and W138 cells (lane D). Six micrograms of RNA was resolved on a formaldehyde gel and transferred to a Genescreen membrane. The blot was probed with a DNA probe corresponding to the coding region of the rat B2 receptor and washed as described in Materials and Methods. The sizes of the major bands are shown in kb.

6.6 - 5.8 -

4-

2-

kb long (McEachern et al., 199 1; Phillips et al., 1992), but the coding domain is only - 1.1 kb in length (McEachern et al., 1991), and much of the length of the message is accounted for by a long 3' untranslated sequence. Although the resolution of nondenaturing sucrose gradients is limited, it is clear that the mRNA encoding the B, receptor is shorter than the B, message by - 2 kb. However, because the protein-encoding region of the B, mRNA is much less than the full length of the RNA, the B,-encoding message may still encode a protein of equal or greater length than the B, polypeptide (364 amino acids in the human).

We used antisense oligonucleotides based on the B, BK receptor sequence to probe the relationship between the coding domains of the two mRNAs. Our initial experiments were aimed at showing that we could inhibit receptor expression specifically with this approach. Our model experiments showed that we could distinguish between the rat 5-HT,, receptor and the B, BK receptor, inhibiting the expression of either of these at will, while sparing expression of the alter- nate receptor by oocytes injected with a mixture of RNAs encoding both receptors. We mixed quite high concentrations of antisense oligodeoxynucleotides in vitro with poly(A)+ RNA or transcripts from cloned cDNAs and injected the mixtures into oocytes. Oligo- nucleotides with conventional phosphodiester bonds survive for only - 30 min in Xenopus oocytes (Cazen-

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1252 M. WEBB ET AL

ave et al., 1987; Woolf et al., 1990), and high concentrations of oligonucleotide have been found necessary to achieve complete inhibition in the Xenopus oocyte system (Woolf et al., 1990). However, the continued presence of the antisense oligonucleotide is not required to maintain inhibition, because the effector mechanism involved is the targeted destruction of RNA/DNA heteroduplexes by an RNaseH-like enzyme (Dash et al., 1987; Shuttleworth and Colman, 1988). The protocol we adopted gave complete inhibition of the targeted receptor, while allowing robust responses by the control receptor. These responses also indicate that the high concentrations of oligonucleotides used in this study did not result in nonspe- cific toxicity to the oocytes.

We used this system to examine the possibility that the mRNAs encoding B, and B, receptors share sequence homology. We made seven oligonucleotides whose sequences were based on either transmembrane or extracellular regions of the human B, receptor sequence. All of these oligonucleotides completely inhibited the expression of the B, receptor, without affecting the expression of the B, receptor. This indicates that, in the regions probed by the oligonucleotides, the two mRNAs are sufficiently different for the oligonucleotides to distinguish between them. The two sequences are probably quite dissimilar, because in the oocyte system, complete matching between the targeted RNA and the oligonucleotide is not required for at least some degree of target destruction (Woolf et al., 1992).

Northern blots of RNA from various tissues probed with a B, BK receptor cDNA showed several bands larger than the 4.0-kb mRNA corresponding to the cloned rat B, sequence, but none smaller (McEachern et al., 199 1). We examined poly(A)+ RNA from W138 cells and found no evidence for an RNA species smaller than 4 kb hybridising to the rat B, probe. This is consistent with the results of our antisense experiments in suggesting that there cannot be a high degree of homology between the B, and B, receptor-encoding mRNAs.

We recently showed that expression of a cloned mouse BK receptor gene in COS cells leads to the expression of both B,-like and B,-like binding sites (McIntyre et al., 1993). If the B,-like site is derived by processing a portion of the “B,” transcript, this obser- vation raises the possibility that B, pharmacology may be generated by two independent molecular mechanisms. If some tissues express appropriate “modifying factors,” they could process a portion of the B, transcript or translation product to give rise to a constitutive level of B, receptor expression. Such a mechanism may underlie the expression of B, receptors by some vascular tissues under normal circum- stances, without apparent insult (Muller-Schwein- itzer, 1988; Ritter et al., 1989). In contrast, the mRNA that we have studied in W138 cells may en-

code a distinct B, receptor that may be the same as the one that is inducible as a result of tissue injury.

Some evidence suggests the existence of a functional heterogeneity that might parallel this hypotheti- cal molecular heterogeneity within the B, receptor subdivision. Thus, Ljungren and Lerner ( 1990) showed that calcium release from acutely isolated osteoblasts was stimulated by des-Arg9-BK, and this response was blocked by Leu’, des-Arg9-BK. Acutely isolated cells responded to BK, but not des-Arg9-BK, by the release of prostaglandin E, and prostacyclin. After prolonged incubation (>24 h), these cells devel- oped the capacity to respond to des-Arg9-BK by the release of prostaglandins, a response that was blocked by Leu’, des-Arg9-BK. Other explanations are possible, but one interpretation of these data is that osteoblasts express two subtypes of B, receptor, one of which is constitutive and mediates calcium release, the other being induced de novo after culture and me- diating prostaglandin production.

Cloning a cDNA sequence representing the B,-type receptor that we have studied in WI38 cells should help to resolve these questions. Our data begin to address the molecular basis for the expression of a distinct B, BK receptor. They also suggest that it will not be possible to clone sequences encoding this receptor by using homology-based methods and that functional expression methods will be required. We are currently pursuing this approach to clone the B, BK receptor from W138 cells.

Acknowledgment: We thank Ms. L. Skidmore for discus- sions of DNA sequences, Dr. s. Chamberlain and Dr. s. Al-Mahdawi for advice on northern blotting, and Dr. S. J. Bevan for help with the oocyte recording and for helpful criticism of the manuscript. We thank Prof. D. Julius for his permission to use the rat 5-HT,, cDNA in these experiments.

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