I) How it started
I was introduced to medical science at the age 10 by my uncle Dag Lundberg who took a blood sample from my finger, stained a slide and let me look in a microscope on my blood cells, the” red ones helping me breathing and the white ones helping me to stay healthy and fight infections”. The cells looked so beautiful and mysterious and stayed as an unforgettable memory. The next step happened at my scholarship experience after student examen at Gustavus Adolphus college, (GAC) Minnesota, USA. I had an icehockey scholarship (and funds from the Kings foundation) to pay for a year studying chemistry and psychology. Unfortunately, I got injured early in the season, so I had a lot of time for studies, which was not so common among the athletes. Then I learned about two Swedish scientists: A Dahlstrom and Kjell Fuxe who had mapped out monoamine containing neuronal systems in the brain that specifically were characterized by noradrenaline, dopamine and serotonin. Increasing evidence suggested that these neurons could have a role in a variety of brain functions like alertness, mood and movement control. Since GAC had a Swedish heritage and I was Swedish, this Swedish science origin discussion became very relevant to me.
After returning to Sweden and starting at Gothenburg medical school in 1973, I trained for a while in Annica Dahlstrom’s lab and learned more about the peripheral nervous system. Furthermore, the courses in Pharmacology and Physiology had excellent teachers like Prof Arvid Carlsson and Prof Björn Folkow lecturing about neuropsychopharmacology and cardiovascular autonomic nervous control, respectively. I became increasingly interested in the mechanisms of chemical nervous system transmission.
II) Discovery of vasoactive intestinal polypeptide (VIP) in cholinergic neurons of exocrine glands
The career changing seminar I attended in fall of 1977 was delivered by Dr Tomas Hökfelt from the Karolinska Institutet (KI), who talked about peptides as mediators of nerve signaling, more specifically substance P(SP), which had been discovered by another Swedish KI scientist (Ulf von Euler). Elegant immunohistochemistry demonstrated the presence of SP in small sensory spinal neurons with central branches into dorsal horn of spinal cord and peripheral ramifications into eg. the skin. This was a novel new field opening up to be studied in more detail about further localization and especially potential functional and pharmacological applications. I had been considering taking a pause in my medical studies to do a PhD thesis (doktorsavhandling) and now this interesting neuropeptide area came in front of my eyes. In addition, the education government decided that top grades (which I had achieved in all subjects) was not as important for medical studies so grades were removed from exams. Consequently, future allocation of medical internships should not be based on grades in courses but on a lottery procedure. Hence, this situation (not being able to “influence “your future) further triggered my desire to add tofurther merits (a PhD) to my CV and to contact Dr Hökfelt and ask if I could come to his lab and learn some new techniques? His lab was already quite full of PhD students and guest scientists, so I had to prove myself to be worthy of a new specific task. Here my new knowledge about peripheral nerves and ganglia came handy. After a test period I was allowed to be a PhD student (without any position or pay) and start to study the peripheral nerve distribution of various neuropeptides (Vasoactive intestinal polypeptide, VIP discovered by V Mutt at KI) and enkephalins, in addition to SP.
At the same time, I wanted to get more functional aspects of peripheral nerves into my thesis program. I therefore went to the Pharmacology department where prof Sune Rosell had interests in pharmacological neuropeptide (SP) antagonists and access to various large animal models. After talking to some science groups there, I finally agreed to work with an experimental clinician Doc Anders Änggård from the ear, nose and throat (ENT) department at the Karolinska hospital. He was an expert in cat microsurgery, autonomic nerve stimulations and blood flow determinations. I now needed to decide a model system for my functional peptide transmission studies.
A fundamental discovery happened in 1979; lumbar sympathetic ganglia in the cat contains classically cholinergic (acetylcholinesterase positive) neurons innervating skin sweat glands of hind paws via the sciatic nerve. We now detected VIP immunoreactivity (IR) in the same neurons, illustrating coexistence of two potentially active chemical messengers VIP and Acetylcholine (ACh). But how could we study the transmitter mechanisms, like mediator release and effects on blood flow and exocrine secretion in a simpler, more easily accessible system? We chose the cat submandibular gland where the same coexistence seemed to exist between acetylcholine and VIP. Further, there was a classic atropine-resistant noncholinergic vasodilatation upon stimulation of the parasympathetic nerves suggesting presence of additional mediator than ACh. In the thesis plan, the following aspects of Dales transmitter criteria were studied:
- 1) How widespread was the coexistence of ACh and VIP -IR in neurons of exocrine glands?
- 2) What was the subcellular storage of ACh and VIP in neurotransmitter storage granules?
- 3) How was the supply of VIP to nerve endings from cell body synthesis?
- 4) How was release of VIP and ACh upon parasympathetic stimulation with differentfrequencies?
- 5) What were the functional responses to VIP or ACh and/or combinations thereof?
- 6) Could the nerve stimulation effect on salivary secretion or increase in blood flowbe modified by atropine or neutralizing VIP antisera?
These aspects were defended in my thesis in 1981 (Fig 1).
Figure 1 VIPergic Cotransmission. From Lundberg Pharmacology Review 1996
VIP and Ach had different subcellular storage and supply. VIP being preferentially stored in large dense cored vesicles, synthesized in the cell body and supplied to terminals by axonal transport. VIP and Ach seemed to have complimentary roles on blood flow and salivary secretion, whereby VIP was primarily a vasodilator, released at high stimulation frequencies, also enhancing cholinergic salivation.
Other labs outside my two thesis tutors also contributed to these activities: Prof V Mutt (VIP peptide supply) Dr J Fahrenkrug (VIP measures, VIP antisera), Prof S Brimijoin (axonal transport).
III) Neuropeptide Y in sympathetic neurons-stress release
Due to a vast collaboration network in Dr Hökfelt’s lab an antiserum to avian pancreatic polypeptide (APP) was tested from Dr Kimmel. This antibody unexpectedly stained noradrenergic (NA) sympathetic neurons. But what was the nature of this immunoreactivity (IR)? The potential solution came later when Dr K Tatemoto in Prof Viktor Mutts lab first isolated another member of the pancreatic polypeptide family (PYY); peptide (P) with N and C terminal tyrosine (Y) from
pig intestine. This peptide had very potent vasoconstrictor activities and inhibited intestinal motility being present in colonic endocrine cells. Soon thereafter a similar peptide; neuropeptide Y (NPY) was isolated from pig brain. We found similar staining as APP-IR of sympathetic nerves with NPY antiserum having a wide distribution in peripheral tissues including heart, vascular system and vas deferens. Interestingly, NPY peptide blocked the APP-IR staining suggesting that the APP antiserum cross reacted with NPY in the tissue. Hence, it was logical to compare the biological activity of NPY. NPY like PYY was a potent vasoconstrictor and in addition inhibited the biphasic contractions of vas deferens induced by electrical stimulation suggesting prejunctional activity on NA and adenosine triphosphate (ATP) transmitter release (Fig 2).
Figure 2. NPYerigic transmission. From Lundberg Pharmacology Review 1996.
NPY caused vasoconstriction also in man (healthy human volunteers) leading to blood pressure elevation (Dr G Ahlborg collaboration). Release of NPY could be measured upon sympathetic nerve stimulation of the pig spleen as model organ, preferentially at high stimulation frequencies. In humans systemic NPY levels rose, indicating release, especially upon heavy physical exercise as a means of activating sympathetic nerves. Furthermore, NPY plasma levels were elevated in patients upon more severe heart failure, a known condition with strong sympathetic nerve activation (Dr J Hulting collaboration). Pharmacological depletion of NA levels by reserpine also reduced tissue NPY levels in certain organs. Furthermore, alpha 2 receptor blockade increased nerve stimulation induced NPY release suggesting that NA was controlling (inhibiting) NPY release. After NA depletion by reserpine, NPY release was enhanced and exceeded resupply with time leading to tissue depletion. As mentioned, reserpine treatment led to long-lasting depletion of NPY in certain tissues including vascular nerves and heart but not in vas deferens. This tissue specific depletion by reserpine of NPY could be prevented by reducing sympathetic nerve activity. After such procedures, in the absence of NA with enhanced NPY release also a long-lasting, presumably NPY mediated, sympathetic nerve stimulation induced vasoconstriction could be demonstrated in several vascular beds (spleen, kidney, skeletal muscle and nasal mucosa) and various species (pig, dog and cat). After NPY receptors were cloned by other groups ,it was determined by NPY peptide fragments that the vasoconstriction was via Y1 receptor in most blood vessels except the pig spleen where Y2 receptor seemed to dominate as for the prejunctional effect in vas deferens. One specific blood vessel which had a slow long-lasting contraction even in control conditions in vitro upon electrical stimulation was guinea pig vena cava where NPY could be involved as transmitter. NPY in addition inhibited the vagal tone of the heart, mimicking the effect of sympathetic nerve stimulation (Potter) presumably via Y2 receptors (Björkman) which may be of relevance for stress induced arrythmias.
IV) Multiple peptides in capsaicin sensitive sensory nerves
After the discovery of SP in sensory nerves, a tachykinin analogue neurokinin A (NKA) was isolated by other groups) from the same peptide precursor. The biological activity of tachykinins were found to be mediated by neurokinin (NK) 1,2 and 3 receptors. The dominant receptor for plasma protein extravasation was NK1, while contraction of visceral smooth muscle including bronchi and urinary tract was NK2 (Fig 3).
Figure 3. Sensory neuron transmission. From Lundberg Pharmacology Review 1996.
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