Danielle Ofri

Characterization of Solubilized Opioid Receptors: Reconstitution and Uncoupling of Guanine Nucleotide-Sensitive Agonist Binding

Abstract: Opioid receptors were solubilized from bovine striatal membranes with the zwitterionic detergent 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate‐(CHAPS). High concentrations of NaCl (0.5–1.0 M) were necessary to ensure optimal yields, which ranged from 40 to 50% of membrane‐bound receptors. This requirement was found to be specific for sodium, with only lithium able to substitute partially, as previously reported for solubilization with digitonin. Opioid antagonists, but not agonists, were able to bind to soluble receptors with high affinity. High‐affinity binding of μ, δ, and κ agonists was reconstituted following polyethylene glycol precipitation and resuspension of CHAPS extract. Evidence is presented suggesting that this is the result of inclusion of receptors in liposomes. Competition and saturation studies indicate that the three opioid receptor types retain their selectivity and that they exist in the reconstituted CHAPS extract in a ratio (50:15:35) identical to that in the membranes. In reconstituted CHAPS extract, as in membranes, μ‐agonist binding was found to be coupled to a guanine nucleotide binding protein (G protein), as demonstrated by the sensitivity of [3H][d‐Ala2, N‐methyl‐Phe4,Gly5‐ol]‐enkephalin ([3H]DAGO) binding to guanosine 5′‐O‐(thiotriphosphate) (GTPγS). In the reconstituted CHAPS extract, complete and irreversible uncoupling by GTPγS was observed, whereas membrane‐bound receptors were uncoupled only partially. Treatment with GTPγS, at concentrations that uncoupled the μ receptors almost completely, resulted in a fourfold decrease in the Bmax of [3H]DAGO binding with a relatively small change in the KD. Competition experiments showed that the Ki of DAGO against [3H]bremazocine was increased 200‐fold. This indicates that the observed decrease in Bmax is due to a reduction in affinity of the uncoupled receptors to a level too low to be measurable, whereas the residual coupled receptors retain high affinity for μ agonists. The methods described should prove useful for opioid receptor purification and reconstitution with G proteins and second messenger systems.

Lesioning of the nucleus basalis of Meynert has differential effects on mu, delta and kappa opioid receptor binding in rat brain: a quantitative autoradiographic study

Opioid receptor binding was investigated in rat brain following lesioning of the nucleus basalis of Meynert (nbM). The nbM, which provides cholinergic input to the cortex, was lesioned unilaterally using ibotenic acid. The efficacy of lesioning was confirmed by the observation of a significant decrease in choline acetyltransferase (ChAT) activity in the ipsilateral prefrontal cortex. The specific laminar and regional distribution of mu, delta and kappa opioid receptor binding was quantitated in various cortical and limbic structures in the rat using autoradiography. Distinct medial to lateral gradients of mu and kappa opioid binding were observed in regions of the cerebral cortex. In the lesioned hemisphere the levels of mu, delta and kappa opioid binding were altered in localized areas of the cerebral cortex and the hippocampus. The direction of these binding changes varied with the opioid receptor type being assessed. Delta opioid binding was increased in the lateral portions of the frontal, occipital, perirhinal and retrosplenial granular cortices. Kappa opioid binding was increased in the lateral portion of the occipital cortex and in the CA3 region of the hippocampus. In contrast, mu opioid binding was decreased in the lateral portions of the frontal, entorhinal and forelimb cortices. These opioid receptor changes are discussed with respect to the interactions between the cholinergic and opioid systems, and relevance of the nbM lesion model to Alzheimers disease.

Neuron overload and the juggling physician

Patients often complain that their doctors don’t listen. Although there are probably a few doctors who truly are tone deaf, most are reasonably empathic human beings, and I wonder why even these doctors seem prey to this criticism. I often wonder whether it is sheer neuron overload on the doctor side that leads to this problem. Sometimes it feels as though my brain is juggling so many competing details, that one stray request from a patient—even one that is quite relevant—might send the delicately balanced three-ring circus tumbling down. One day, I tried to work out how many details a doctor needs to keep spinning in her head in order to do a satisfactory job, by calculating how many thoughts I have to juggle in a typical offi ce visit. Mrs Osorio is a 56-year-old woman in my practice. She is somewhat overweight. She has reasonably well-controlled diabetes and hypertension. Her cholesterol is on the high side but she doesn’t take any medications for this. She doesn’t exercise as much as she should, and her last DEXA scan showed some thinning of her bones. She describes her life as stressful, although she’s been good about keeping her appointments and getting her blood tests. She’s generally healthy, someone who’d probably be described as an average patient in a medical practice, not excessively complicated. Here are the thoughts that run through my head as I proceed through our 20-min consultation.

Public health and the muse

At first glance, it might seem odd that a public health journal would initiate a section about arts and humanities. Public health, after all, deals with populations; it eschews the individual except as it forms one of a group. The creative arts, however, deal almost exclusively with individuals. Literature, in particular, always has a protagonist, and the protagonist is never ‘alcoholics with pancreatitis,’ ‘female prisoners receiving hepatitis B vaccination,’ ‘South Asians with cardiovascular risk factors,’ ‘UK asylum seekers with infectious disease,’ or ‘teenaged asthmatic smokers.’ A protagonist is an individual. Madame Bovary, Huckleberry Finn, Jay Gatsby, Pip, Hamlet, Odysseus, Harry Potter, Holden Caulfield, Captain Ahab, Anna Karenina, Sherlock Holmes and Jean Valjean are individuals, not populations. What happens to each is entirely unique. There is nothing in their characters that is ‘applicable’ to larger populations; they define individualism. Our pleasure in reading these novels is the exhilaration of being swept up in the singular journeys of these remarkable individuals. As an academic internist, I teach medical students and junior doctors in both the inpatient and outpatient settings. I have often been disappointed in how easily the house staff lose the trees for the forest. Too often, they take a ‘population’ approach to their patients, though not exactly in the public health sense, more in a category approach: Chest pain patient1⁄4 telemetry, serial enzymes, echo, stress. Pneumonia patient1⁄4 X-ray, IV ceftriaxone plus azithro. Altered mental status patient1⁄4 CT, LP, pan-culture, broad-spectrum antibiotics. A few years ago I began to supplement rounds with readings from classic literature or our own Bellevue Literary Review. I always chose stories, essays, or poems that specifically highlighted the individual. I wanted to use literature’s greatest strength—the uniqueness of the protagonist—to remind junior doctors that because each patient’s chest pain occurs in a different life that each chest-pain story is, by definition, exceptional. Up until the invitation to write this essay, however, I had never thought to look at literature from a public health perspective. Like most internists, my gaze is locked only on the one patient in front of me, and then the next one, and then the next one. There is rarely time to pick up one’s head and consider the wider medical perspective. And like most writers, my gaze is locked only on the one story in front of me. Therefore, I was forced to pause and think about how I might pull together literature and public health. After reading some of the current literature of public health, one aspect resonated most strongly to me as a practicing clinician—the understanding of the social context of disease. Social context is a broad palette. In the second paragraph of this essay, I listed some of the most memorable protagonists in literature. Each is highly individual and unique. But each inhabits a complex, carefully wrought environment. Most are defined by, or defined against, their respective social contexts. Perhaps this is one way in which the humanities can be applicable to public health. Since title of this section of the journal was to be ‘Chekhov’s Corner,’ I felt duty-bound to revisit the master. I pushed aside my medical journals and dusted off my volume of Chekhov stories. Reading them again was like returning to the motley but familiar assemblage of eccentrics and ordinary folks living in a muted corner of my history. Indeed, this New York Public Library edition was entitled ‘Motley Tales and a Play.’ (As a writer, I am deadly envious of anyone who manages to get a blurb from Tolstoy on the back cover of his book.) I read through the stories, skeptical that I would find anything that would relate to public health. Each story is about a very particular individual and the very particular circumstances into which he or she is plopped by a dexterous literary creator. The story of the sexton’s wife who is mesmerized by the face of the mail carrier lost in a

Abortion: the view from both sides of the street

www.thelancet.com Vol 376 July 31, 2010 321 12th & Delaware Directed by Rachel Grady and Heidi Ewing. HBO Films, 2010. Broadcast on HBO on Aug 2 and Aug 5, 2010. See http://www. hbo.com/documentaries/12thand-delaware/index.html A dispassionate discourse on the abortion wars in the USA? Not something that seems possible, at least in the current polarised culture. Almost by defi nition, any analysis of the politics and practice of abortion is heavily partisan. Even the medical world—the last bastion of any possible objectivity—has been overlaid with politics. Outside of major urban medical centres, pregnancy termination has been sliced off from the greater fi eld of obstetrics and gynaecology, isolated in freestanding abortion clinics that have become the last hope for desperate women and the target for desperate protesters. Into this fray comes the documentary 12th & Delaware, a quiet movie that seeks to illuminate rather than bully. In 1990, a husband and wife team—Candace and Albert—opened the Women’s World health clinic on the corner of 12th and Delaware in a nondescript Florida neighbourhood. Motivated by their desire to help women obtain abortions, Candace provides counselling to the patients and Albert shuttles the doctors, who remain hidden under sheets in his car so that they will not be recognised, trailed, or assassinated. In 1999, the house across the street went up for sale. Within 24 hours it had been purchased by a pro-life group that opened the Pregnancy Care Center. The clinic is supported by the Catholic Church and seeks to persuade women not to have abortions. 12th & Delaware shows life in the two clinics with little fanfare and no commentary. There is no narration or punditry. Other than a rare line of text setting the stage, we hardly see the hand of directors Rachel Grady and Heidi Ewing. First we follow Anne, a counsellor at the Pregnancy Care Center. We see her talking to a 15-year-old pregnant teenager. When she estimates gestational age, she hands the girl a plastic fetus from a collection of all sizes. “Go ahead and hold it”, she says, urging her to take it in her hands. “This is what your baby is like.” Then there is the free ultrasound. Anne encourages another woman and her boyfriend to look at “their baby”, to see the heartbeat. The technician types “Hi Mommy, Hi Daddy” into the machine so that the words appear on the ultrasound picture that is given to the couple.

Adding Spice to the Slog: Humanities in Medical Training.

Writing from personal experience, physician and author Danielle Ofri asks what evidence is needed to justify trying to humanize medical training via the power of literature.

Pharma in the jungle

How to make pharmaceutical R&D worthy of James Bond-like drama? It’s not easy, but Ann Patchett gamely tries. An American drug company based in the dull stretches of Minnesota is racing to develop the holy grail of fertility drugs—a simple pill to allow women to get pregnant at any age. The stockholders are rubbing their palms rapaciously at the mere thought. Deep in the far reaches of the Amazon rainforest along the banks of the Rio Negro, the Lakashi tribe have been living quietly, procreating without fanfare well into their eighth decade. An elusive but brilliant scientist, Dr Annick Swenson, has discovered that these women gnaw on the bark of a rare tree deep in the jungle, and that the bark imparts fertile longevity that would make an IVF clinic blush. However, Dr Swenson has been taking her own sweet time on the research and the pharmaceutical company is growing impatient—especially as she has eschewed all forms of contact. They don’t even know exactly where she is. So the company sends a fellow scientist, Anders Eckman, to track her down and report on the state of drug development. Unfortunately, native Minnesotans don’t do well in the Amazon, and within weeks Eckman is dead of a febrile illness. Thus his lab partner—our heroine— is dispatched to uncover the details of his death. Marina Singh is a loner pharmacologist, having quit her obstetrics–gynaecology residency at Johns Hopkins after a horrendous medical error, oddly enough under the auspices of the imperious department chair, none other than Dr Annick Swenson. As is often the case, the senior physician abandoned the junior physician in the face of medicolegal calamity, and we know who was left to face the fl ames. Marina suff ers the various insults of the tropics during her hunt for the elusive Dr Swenson—fl otillas of insects, lost luggage, venomous snakes, psychogenic side-eff ects of antimalarials, intermittent fevers, generalised disorientation. Despite carefully crafted prose, none of these misadventures have the sizzle of Joseph Conrad’s Heart of Darkness, which is manifestly beating in the background of Patchett’s novel.

A “difficult” patient's journey

2074 www.thelancet.com Vol 377 June 18, 2011 My Imaginary Illness: A Journey into Uncertainty and Prejudice in Medical Diagnosis Chloë G K Atkins. ILR Press/Cornell University Press, 2011. Pp 248. US

The Debilitated Muse: Poetry in the Face of Illness

27·95. ISBN 9780801448874 Book A “diffi cult” patient’s journey Chloë Atkins is the type of patient that every doctor dreads—presenting with a plethora of symptoms that don’t off er any obvious medical explanation. There are multitudes of such patients in a general practitioner’s roster and most, thankfully, will not turn out to have a serious illness. But there are a few who do, and as Atkins’ book points out, this can be a harrowing experience. Atkins turned out to have an atypical presentation of myasthenia gravis, and it took nearly 20 years to get this sorted out. As doctor after doctor was unable to fi nd a diagnosis, her syndrome was labelled as psychosomatic, and Atkins chronicles the increasing hostility of the medical profession towards her. The book presents the stark reality of how medicine falters when faced with uncertainty. Doctors notoriously resent uncertainty, and this becomes quickly manifest in the doctor–patient relationship. We all have those “diffi cult” patients, and Atkins bluntly puts herself in that category, candidly admitting that she “burned people out”. That doctors are uncomfortable with ambiguity shouldn’t come as any surprise, based on our education that is grounded in the seeming solidity of facts. But there is also the element of fear: which of these many patients with vague and varied symptoms harbours a serious illness? This needle-in-the-haystack pressure is compounded by the unfortunate reality of short outpatient visits (or overfl owing inpatient wards). It is impossible for even brilliant doctors to discern complicated, mysterious illnesses in 15 minutes or less. By now, most physicians have abandoned the blatant “it’s all in your head” fall-back. Most of us feel that the chronic pain syndromes, irritable bowels, and fi bromyalgias do indeed have a biological basis, even if poorly understood. But stress does wreak additional havoc on these illnesses, so it’s not unreasonable to work to ameliorate this. Most of us view these illnesses as “syndromes” of some sort, and try to manage both the biological and psychological sides. Atkins is furious at the medical profession for not diagnosing her illness earlier and for not taking her symptoms seriously (she calls the book a “justice narrative”). Her anger is entirely understandable. But in reading the book, I had to honestly wonder whether her condition was actually possible to diagnose. Even the clinicians who took her seriously were stymied by uncharacterisable symptoms and confl icting test results. Even if every doctor had been the paragon of attentiveness, respect, and doggedness, it is quite likely that this rare disease with its atypical presentation would have been missed. Atkins’ argument that her doctors’ attitudes were the cause of her misdiagnosis is in my view the most tangible shortcoming of the book. Certainly, these attitudes are shameful, harmful, and in need of addressing, but it’s not possible to draw the conclusion of causality in such a complex and inscrutable case. This book is the fi rst in a series entitled “How Patients Think”, a complement—or retaliation, depending on your bias—to Jerome Groopman’s How Doctors Think. Patients, however, are much more heterogeneous than doctors, so such a series will likely off er a host of individual experiences that may or may not be universal. But this still has immense value. Doctors easily fall into the trap of categorising patients, and we need reminding of their individuality. My Imaginary Illness may not have the philosophical or literary reach of, say, Anatole Broyard’s Intoxicated by My Illness, but it is instructive nevertheless. Atkins’ case of a rare illness is itself not generalisable, however the fl aws in the system that she illuminates certainly are.

Reconstitution in liposomes of a μ-opioid binding protein purified to homogeneity from bovine striatal membranes

Poetry is a supremely sensory art, both in the imagining and in the writing. What happens when the poet faces illness? How is the poetry affected by alterations of the body and mind? This paper examines the poetry of several writers afflicted by physical illness—poets of great renown and poets who might be classified as “emerging voices,” in order to explore the interplay between creativity and corporeal vulnerability.